Methods for producing crosslinked flavonoid hydrogels

ABSTRACT

There is provided methods for producing a hydrogel comprising conjugates of a hydrogel forming agent and a flavonoid including a method for producing a hydrogel that is capable of adhesion of cells and which comprises enzymatically cross-linked conjugates of a hydrogel forming agent and a flavonoid. There is also provided a method for producing a hydrogel comprising conjugates of a hydrogel forming agent and a flavonoid without the addition of an exogenous peroxide or peroxidase or without the addition of an exogenous peroxide. Hydrogels produced by such methods and methods of using the hydrogels are also described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/970,432, filed Aug. 19, 2013, which is a divisional of U.S. patentapplication Ser. No. 13/375,097, filed Nov. 29, 2011, issued as U.S.Pat. No. 8,541,016 on Sep. 24, 2013, which is a U.S. National Stageapplication based on International Application No. PCT/SG2010/000185,filed May 17, 2010, which claims benefit of, and priority from, U.S.provisional patent application No. 61/213,331 filed on May 29, 2009, thecontents of each of which are fully incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates generally to hydrogels comprisingconjugates of a hydrogel forming agent and a flavonoid and methods forpreparing and using the hydrogels.

BACKGROUND OF THE INVENTION

Flavonoids are one of the most numerous and best-studied groups of plantpolyphenols. The flavonoids consist of a large group of low-molecularweight polyphenolic substances naturally occurring in fruits andvegetables, and are an integral part of the human diet. Dried green tealeaves can contain as much as 30% flavonoids by weight, including a highpercentage of flavonoids known as catechins (flavan-3-ol derivatives orcatechin-based flavonoids), including (−)-epicatechin,(−)-epigallocatechin, (+)-catechin, (−)-epicatechin gallate and(−)-epigallocatechin gallate.

In recent years, these green tea catechins have attracted much attentionbecause they have been recognized to have biological and pharmacologicalproperties, including anti-bacterial, anti-neoplastic, anti-thrombotic,vasodilatory, anti-oxidant, anti-mutagenic, anti-carcinogenic,anti-hypercholesterolemic, anti-viral and anti-inflammatory properties,which have been demonstrated in numerous human, animal and in vitrostudies [30-32]. These biological and pharmacological properties arepotentially beneficial in preventing diseases and protecting thestability of the genome. Many of the beneficial effects of catechins arethought to be linked to the antioxidant actions of the catechins [33].Among the catechins, (−)-epigallocatechin gallate (EGCG), which is amajor component of green tea, is thought to have the highest activity,possibly due to the trihydroxy B ring and the gallate ester moiety atthe C3 position [34-38]. EGCG has been recognized to have biochemicaland pharmaceutical effects including anti-oxidant, anti-carcinogenic,and anti-inflammatory properties [5-7]. EGCG is known to inhibit a vastarray of biomedically relevant molecular targets and disease-relatedcellular process [8] consequently leading to the induction of apoptosis,inhibition of tumour cell growth, and inhibition of angiogenesis [9].These beneficial bioactivities are attributed mostly to the strongbinding ability of EGCG to many biological molecules, including peptidesand proteins, which affect various enzyme activities and signaltransduction pathways [10]. EGCG is also known as a potent inhibitor ofmatrix metalloproteinase (MMP) gelatinases [11] which play a crucialrole in tumour metastasis.

Studies have found that a high EGCG dosage is required to exert thesetherapeutic effects. However, high micromolar concentrations of EGCG cannot practically be achieved from simple dietary intake [8]. In general,the activity half-life of flavonoids is limited to a few hours insidethe body; metabolism of these compounds has not yet been established.Despite the favourable anti-oxidation and anti-cancer properties of thecatechins such as EGCG, it is impractical to achieve a therapeutic levelof this compound in the body by directly ingesting a large amount ofgreen tea, due to the inherent volume constraint. That is, in order toobtain a therapeutic or pharmacological benefit from flavonoids throughdiet alone, it would be necessary to ingest an amount of food andbeverage that is larger than is practical to consume. Moreover,pro-oxidant activity has been reported for several flavonoids includingEGCG, making ingesting crude green tea directly a less effective meansof delivering EGCG [39-41]

On the other hand, a relatively high-molecular fraction of extractedplant polyphenols (procyanidins) and synthetically oligomerized(+)-catechin and rutin have been reported to exhibit enhancedphysiological properties such as antioxidant and anti-carcinogenicactivity compared to low-molecular weight flavonoids, [42-46] withoutpro-oxidant effects [47,48]. However, neither naturally occurring norsynthesized high molecular weight flavonoids are expected to be absorbedand transported to other tissues after ingestion, since these compoundsare typically large, form strong complexes with proteins and areresistant to degradation [49].

In cases of flavonoids consumed via oral intake of foods and beverages,the flavonoids may play a role as antioxidants to protect the digestivetract from oxidative damage during digestion. However, flavonoids can beexpected to remain only in the digestive tract and thus their beneficialphysiological activities are not likely to be utilized in other tissues.Moreover, their strong hydrophobicity as well as their tendency to formcomplexes with proteins makes parenteral delivery of these compoundsdifficult.

SUMMARY OF INVENTION

There is presently provided methods for producing a hydrogel comprisingconjugates of a hydrogel forming agent and a flavonoid. In oneembodiment, there is provided a method for producing a hydrogel capableof adhesion of cells and methods for forming such a hydrogel. Thehydrogel is formed from conjugates of a hydrogel forming agent, forexample a polymer, and a flavonoid, for example a catechin-basedflavonoid, for example epigallocatechin gallate, using a sufficientamount of a peroxidase enzyme, including horseradish peroxidase, toinduce, in the presence of a peroxide, for example hydrogen peroxide,sufficient enzymatic cross-linking in the hydrogel to produce a hydrogelcapable of adhesion of cells.

In another embodiment, there is provided methods for producing ahydrogel comprising conjugates of a hydrogel forming agent and aflavonoid without the addition of an exogenous peroxide or peroxidase orwithout the addition of exogenous peroxide.

There are also provided methods for adhering a cell to a hydrogel,including for inhibiting proliferation of a cancer cell and forproliferating a non-cancer cells and methods of delivering a flavonoidto a cell.

Due to the numerous beneficial biological and pharmacological propertiesof flavonoids, the present hydrogels may provide a number of therapeuticproperties useful for treating a variety of diseases, disorders andconditions including bacterial infections, viral infections, vasculardisease, high cholesterol and inflammation.

For example, due to the anti-neoplastic, anti-angiogenesis,anti-oxidant, anti-mutagenic, and anti-carcinogenic properties offlavonoids, the hydrogels described herein may be useful for thetreatment of cancer. Conveniently, additional anti-cancer agents may beincluded within the hydrogel to increase the anti-cancer effect of thehydrogels. The hydrogels may be used in vivo to reduce or preventproliferation of cancer cells while permitting the survival andproliferation of non-cancer cells. By using a biodegradable agent suchas a biodegradable polymer as the hydrogel forming agent, the flavonoidand any additional anti-cancer agent included in the hydrogel can begradually released as the polymer is degraded and absorbed within thebody of a subject.

Thus in one aspect, there is provided a method for producing a hydrogelthat is capable of adhesion of cells and which comprises enzymaticallycross-linked conjugates of a hydrogel forming agent and a flavonoid, themethod comprising combining from about 0.1 mg/ml to about 500 mg/ml ofconjugates of the hydrogel forming agent and the flavonoid; from about0.001 mM to about 50 mM peroxide; and from about 0.001 units/ml to about10 units/ml peroxidase; thereby producing the hydrogel. In a particularembodiment, the method comprises combining from about 1 mg/ml to about100 mg/ml of conjugates of the hydrogel forming agent and the flavonoid;from about 0.01 mM to about 5 mM peroxide; and from about 0.01 units/mlto about 10 units/ml peroxidase.

In particular embodiments, the peroxidase may be horseradish peroxidase,the peroxide may be hydrogen peroxide, the flavonoid may be acatechin-based flavonoid. In one embodiment, the flavonoid isepigallocatechin gallate.

In particular embodiments, the hydrogel forming agent is a polymer. Inone embodiment, the hydrogel forming agent is hyaluronic acid.

In one embodiment, the method for producing a hydrogel that is capableof adhesion of cells and which comprises enzymatically cross-linkedconjugates of a hydrogel forming agent and a flavonoid, furthercomprises combining a bioactive agent with said conjugates, peroxide andperoxidase. In one embodiment, the bioactive agent may be an anti-canceragent, including, for example, herceptin.

In another aspect, there is provided a hydrogel capable of adhesion ofcells and which comprises enzymatically cross-linked conjugates of ahydrogel forming agent and a flavonoid, the hydrogel produced by amethod comprising combining: from about 0.1 mg/ml to about 500 mg/ml ofconjugates of the hydrogel forming agent and the flavonoid; from about0.001 mM to about 50 mM peroxide; and from about 0.001 units/ml to about10 units/ml peroxidase.

In a particular embodiment the hydrogel capable of adhesion of cells andwhich comprises enzymatically cross-linked conjugates of a hydrogelforming agent and a flavonoid is produced by combining from about 1mg/ml to about 100 mg/ml of conjugates of the hydrogel forming agent andthe flavonoid; from about 0.01 mM to about 5 mM peroxide; and from about0.01 units/ml to about 10 units/ml peroxidase;

In particular embodiments, the peroxidase may be horseradish peroxidase,the peroxide may be hydrogen peroxide, the flavonoid may be acatechin-based flavonoid. In one embodiment, the flavonoid isepigallocatechin gallate.

In particular embodiments, the hydrogel forming agent is a polymer. Inone embodiment, the hydrogel forming agent is hyaluronic acid.

In one embodiment, the hydrogel that is capable of adhesion of cells andwhich comprises enzymatically cross-linked conjugates of a hydrogelforming agent and a flavonoid, further comprises a bioactive agent. Inone embodiment, the bioactive agent may be an anti-cancer agent,including, for example, herceptin.

In yet another aspect, there is provided a method for adhering a cell toa hydrogel, the method comprising contacting the cell with a hydrogelthat is capable of adhesion of cells and which comprises enzymaticallycross-linked conjugates of a hydrogel forming agent and a flavonoid, asdescribed herein.

In different embodiments, the cell may be a cancer cell, whereinproliferation of the cancer cell is inhibited or a non-cancer cellwherein the non-cancer cell is proliferated. In different embodimentsthe cell may be in vitro or in vivo.

In particular embodiments, the method for adhering a cell to a hydrogeldescribed herein comprises administering to a subject the hydrogelcomprising an effective amount of the conjugates for the treatment ofcancer.

In another aspect, there is provided a method for producing a hydrogelcomprising conjugates of a hydrogel forming agent and a flavonoid, themethod comprising combining the conjugates in a solution in the absenceof an exogenously added peroxide and in the absence of a peroxidase. Inparticular embodiments, the method comprises controlling the gelationrate of the hydrogel by modifying the pH of the solution. In oneembodiment, the pH is modified between 3 and 10. In another embodiment,the pH is modified between 6 and 8. In another particular embodiment,the method comprises adding catalase to the solution.

In yet another aspect, there is provided a method for producing ahydrogel comprising conjugates of a hydrogel forming agent and aflavonoid, the method comprising combining the conjugates and aperoxidase in a solution in the absence of an exogenously addedperoxide.

In particular embodiments of the method for producing a hydrogelcomprising conjugates of a hydrogel forming agent and a flavonoid, themethod comprising combining the conjugates in a solution in the absenceof an exogenously added peroxide and in the absence of a peroxidase andthe method for producing a hydrogel comprising conjugates of a hydrogelforming agent and a flavonoid, the method comprising combining theconjugates and a peroxidase in a solution in the absence of anexogenously added peroxide. In different embodiments, the flavonoid maybe a catechin-based flavonoid, including, for example, epigallocatechingallate and the hydrogel forming agent may be a polymer, including, forexample, hyaluronic acid. In particular embodiments, the concentrationof the conjugates in the solution may from about 0.1 mg/ml to about 500mg/ml. In one embodiment, the concentration of the conjugates in thesolution is from about 1 mg/ml to about 100 mg/ml. In still otherparticular embodiments, the methods may further comprise combining abioactive agent with the conjugates in the solution. The bioactive agentmay be an anti-cancer agent, including, for example, herceptin.

In another aspect, there is provided a hydrogel comprising conjugates ofa hydrogel forming agent and a flavonoid, the hydrogel produced by themethod for producing a hydrogel comprising conjugates of a hydrogelforming agent and a flavonoid, the method comprising combining theconjugates in a solution in the absence of an exogenously added peroxideand in the absence of a peroxidase, described herein, or the method forproducing a hydrogel comprising conjugates of a hydrogel forming agentand a flavonoid, the method comprising combining the conjugates and aperoxidase in a solution in the absence of an exogenously addedperoxide, described herein.

In another aspect, there is provided a method for delivering a flavonoidto a cell, the method comprising contacting the hydrogel described inthe preceding paragraph with the cell.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention:

FIG. 1. Schematic illustration of versatile HA-EGCG hydrogel for cancertherapy.

FIG. 2. Cell adhesion. HT-1080 cells cultured on (a) plastic well plate;(b) Gtn-HPA; (c) HA-Tyr; (d) HA-Tyr/Gtn-HPA (20:80 w/w); (e)oxygenic-cross-linked HA-EGCG; and (f) enzymatic cross-linked HA-EGCG.

FIG. 3. The effect of HRP concentration on cell adhesion and spreading.(a) HT-1080 cells; and (b) HepG2 cells cultured on HA-EGCG hydrogelenzymatically cross-linked by (i) 0; (ii) 2.3; (iii) 3.2; (iv) 4.1; and(v) 4.9 units/ml of horse radish peroxidase (HRP).

FIG. 4. Cell spreading. Cell morphology was analyzed on the projectedcell area as a function of HRP concentration in substrate after 24 h, 96h and 144 h incubation. Cell spreading of (a) HT-1080; (b) Hep G2; and(c) HFF-1 on enzymatically cross-linked HA-EGCG hydrogel. Cells culturedon plastic well plate were used as a control.

FIG. 5. Cell proliferation and morphology of HT-1080 cells. HT-1080cells were cultured on HA-EGCG hydrogel cross-linked using HRP (4.1units/ml) for the duration of (a) 24, (b) 48, (c) 96, and (d) 144 h.HA-EGCG hydrogel induced growth inhibition and the morphological changein HT-1080 cells. The morphology of cells was observed underphase-contrast microscopy with 200× power.

FIG. 6. Cell proliferation and morphology of Hep G2 cells. Hep G2 cellswere cultured on HA-EGCG hydrogel cross-linked by HRP (4.1 units/ml) forthe duration of (a) 24, (b) 48, (c) 96, and (d) 144 h. HA-EGCG hydrogelinduced growth inhibition and the morphological change in Hep G2 cells.The morphology of cells was observed under phase-contrast microscopywith 200× power.

FIG. 7. Cell proliferation and morphology of HFF-1 cells. HFF-1 cellswere cultured on HA-EGCG hydrogel cross-linked by HRP (4.1 units/ml) forthe duration of (a) 24, (b) 48, (c) 96, and (d) 144 h. HA-EGCG hydrogelenables proliferation of human fibroblast cells. The morphology of cellswas observed under phase-contrast microscopy with 200× power.

FIG. 8. AlamarBlue® assay for cell proliferation. Cell proliferation of(a) HT-1080 (b) HFF-1 assessed by AlamarBlue® assay. Cells were culturedon HA-Tyr/Gtn-HPA mixed hydrogel (A) and HA-EGCG hydrogel (o).

FIG. 9. DNA fragmentation assay for apoptosis. Nucleus morphology ofcells cultured on HA-EGCG hydrogel after 120 h. The nuclei of Hep G2cells were visualized by DAPI staining in blue (light grey regions inblack and white image)(left image (a)). DNA fragmentation was observedusing a fluorescence microscope with 200× power. Photo on the right isoverlaid image of fluorescent probe and phase contrast (b).

FIG. 10. Cell invasion assay. Diff Quik staining of migrated HT-1080cells in Matrigel™ invasion chamber (left image (a)) and HA-ECGChydrogel invasion chamber (right image (b)).

FIG. 11. Tumour growth inhibition of human breast BT474 cancer in mice.Mice were treated with control (∘), HA-EGCG hydrogel (Δ), herceptin (□)or herceptin loaded HA-EGCG hydrogel (▴).

FIG. 12. Formation of HA-EGCG hydrogel through air-autoxidation atdifferent pH. The gelation time of HA-EGCG hydrogels formed byair-autoxidation was reduced with increased pH.

FIG. 13. Acceleration of HA-EGCG hydrogels through air-autoxidation bythe addition of catalase. The gelation time decreased with increasedcatalase concentration.

FIG. 14. Production of H₂O₂ by HA-EGCG conjugates. H₂O₂ generated byHA-EGCG during air-autoxidation increased with time and was observed inthe micromolar range.

FIG. 15. Formation of HA-EGCG hydrogels by HRP-mediated crosslinkingreaction without the addition of exogenous H₂O₂. At pH 7.4, the gelationtime of HA-EGCG decreased with increased concentrations of HRPconcentration.

DETAILED DESCRIPTION

Flavonoids are known to have a variety of beneficial properties,including anti-bacterial, anti-neoplastic, anti-thrombotic,vasodilatory, anti-oxidant, anti-mutagenic, anti-carcinogenic,anti-hypercholesterolemic, anti-viral and anti-inflammatory effects. Toincrease the availability of beneficial flavonoid compounds, flavonoidsmay be conjugated to various agents allowing for modification of thephysical properties of the flavonoid and augmenting the biological andpharmacological properties of the flavonoid without disrupting thepolyphenol structure of the flavonoid.

Conjugation of an agent to a flavonoid can provide a composition that issuitable for administration to a subject by incorporating the flavonoidinto a particular vehicle formed with the conjugate, and thus can allowfor administration of higher concentrations of flavonoids than can beobtained through diet.

Delivery vehicles comprising such conjugates were previously describedin published international application WO 2006/124000 and published USapplication 2008/102052, the contents of which are herein fullyincorporated by reference. Hydrogels comprised of conjugates of ahydrogel forming agent and a flavonoid, referred to herein as flavonoidconjugate hydrogels, are one form of delivery vehicle that can provideimproved effective delivery of flavonoids to particular targeted sitesin the body.

Hydrogels are highly hydrated suspensions comprised of a cross-linkednetwork of hydrophilic molecules dispersed within water. The structuralarrangement of a hydrogel derives from cross-links formed between themolecules by various chemical and physical bonds [51]. Physicalcrosslinking utilizes physical interactions between polymers to formcrosslinks, such as such as ionic, substrate-ligand or hydrophobicinteractions. Chemically crosslinked hydrogels, which are usually formedby a Michael-type addition reaction, disulfide bond formation andaldehyde-mediated crosslinking, have improved mechanical properties andstability in comparison to physically crosslinked hydrogels.

Hydrogels have been widely used for drug delivery and as scaffolds intissue engineering. Particularly, injectable and biodegradable hydrogelsystems have received much attention because of the elimination ofsurgical implantation and retrieval. Hydrogels are a convenient form ofdelivery vehicle as the cross-linked structure allows for controlled andsustained release of therapeutic agents as well as protection of suchagents from degradation by hostile environmental factors such as enzymesand low pH [50]. Recently, gelatin hydrogel and cholesterol-bearingpullulan hydrogel have been developed as carriers for sustainedimmunoprotein release and suppression of tumor growth in vivo viasubcutaneous injection administration [2, 3].

Typically, unmodified hydrogels do not promote cellular adhesion due totheir hydrophilic nature [51]. To date, modification of hydrogels withcell binding peptides such as RGD peptides, or proteins such as collagenor gelatin, has been necessary in order to achieve cell adhesion [19,20].

The present inventors have made the surprising discovery that cellsadhere to hydrogels comprised of conjugates of flavonoids and hydrogelforming agents, referred to herein as flavonoid conjugates, and formedby enzymatic cross-linking of the flavonoid conjugates, without the needfor modification of the hydrogel with additional components such as cellbinding peptides or proteins.

“Enzymatic cross-linking”, also referred to herein as “enzyme-catalyzedcross-linking” or “peroxidase-catalyzed cross-linking”, refers to theformation of cross-links between flavonoid moieties, particularlyoxygen-oxygen cross-links, as the result of an oxidation reactioncatalysed by a peroxidase enzyme. This is in contrast to “oxygeniccross-linking”, which refers to the formation of cross-linking byoxygenic-oxidation, without any enzyme catalysis, described below.

The term “enzymatically cross-linked” is used herein to describecross-linking between two or more flavonoid moieties or flavonoidconjugates formed by enzymatic cross-linking, that is cross-linkingformed as a result of an oxidation reaction catalaysed by a peroxidaseenzyme.

Without being limited to any particular theory, it appears that aparticular flavonoid moiety may be cross-linked to more than one otherflavonoid moiety via enzymatic cross-linking, which may result in theformation of flavonoid oligomers within the formed hydrogel. As usedherein, reference to flavonoid oligomers that are the result of theenzymatic cross-linking refers to two or more flavonoid moietiesconnected via oxygen-oxygen linkages catalysed by a peroxidase enzyme inthe presence of peroxide. In the absence of enzymatic cross-linking,flavonoid conjugates undergo oxygenic-oxidation (also known asauto-oxidation) to produce superoxide radicals and flavonoid radicalswith unpaired electrons delocalized around the B ring. A chain reactionis propagated by the reaction of the superoxide radicals with theflavonoid radicals resulting in oxygenic cross-linking of the flavonoidsto form flavonoid dimers and H₂O₂ [9, 21].

In contrast, in the presence of a peroxidase enzyme, such as horseradishperoxidase, and peroxide, the flavonoid moieties within the flavonoidconjugates are cross-linked by enzymatic oxidation (i.e. enzymaticcross-linking). Without being limited to any particular theory, theenzymatic cross-linking process may involve two successive steps: first,the peroxidase is oxidized by peroxide, to form an intermediate; andsecond, this intermediate then oxidizes a phenolic hydroxyl group on theflavonoid, resulting in the formation of flavonoid oligomers viaoxygen-oxygen cross-linking of the phenol groups and the formation of aflavonoid conjugate hydrogel.

As oxygenic cross-linking and enzymatic cross-linking may occursimultaneously, a flavonoid conjugate hydrogel synthesized using across-linking peroxidase enzyme may consist of a complex mixture offlavonoid dimers and oligomers. However, enzymatic oxidation occurs morequickly than oxygenic oxidation. Thus, with increasing amounts ofperioxidase enzyme, an increasing amount of cross-links between theflavonoid moieties within the hydrogel should be formed by enzymaticcross-linking rather than by oxygenic cross-linking. With a sufficientamount of peroxidase, the available flavonoids may be exhausted by theenzymatic cross-linking thus inhibiting the formation of dimers byoxygenic oxidation of the flavonoids.

The inventors have found that the concentration of peroxidase enzyme,such as horseradish peroxidase, and thus the proportion of enzymaticcross-linking, used in synthesizing the hydrogel affects cell adhesionto the flavonoid conjugate hydrogel. Cell adhesion to a hydrogel formedwith sufficient amount of enzymatic cross-linked flavonoid conjugates ispossible. In contrast, cells do not adhere to a flavonoid conjugatehydrogel formed only with oxygenic cross-linked flavonoid conjugates.

The ability of a flavonoid conjugate hydrogel to adhere cells thus canbe expected to depend on the extent of the enzymatic cross-linkingbetween flavonoid moieties, and accordingly, the amount of peroxidaseenzyme, for example horseradish peroxidase (HRP), used in the formationof the flavonoid conjugate hydrogel.

Cell adhesion to the flavonoid conjugate hydrogel described herein mayprovide improved delivery of flavonoids to a cell. When adhered to thehydrogel, a cell is retained in close proximity to the flavonoidconjugates and any other therapeutic agents contained in and releasedfrom the hydrogel. Such “cell trapping” may improve the efficiency andsuccess of flavonoids and additional therapeutic agents reaching thecell.

Thus, there is presently provided, a method for adhering a cell to ahydrogel by contacting the cell to a hydrogel comprising enzymaticallycross-linked conjugates of a hydrogel forming agent and a flavonoid. Amethod for producing such a hydrogel contemplates the use of conjugatesof a hydrogel forming agent and a flavonoid, peroxide and peroxidase,all at concentrations sufficient to enzymatically cross-link theflavonoids and produce a hydrogel capable of adhesion of cells. Themethod may comprise combining from about 0.1 mg/ml to about 500 mg/ml ofconjugates of a hydrogel forming agent and a flavonoid; from about 0.001mM to about 50 mM peroxide; and from about 0.001 units/ml to about 10units/ml peroxidase; thereby producing a hydrogel that is capable ofadhesion of cells and which comprises enzymatically cross-linkedconjugates of a hydrogel forming agent and a flavonoid. In anotherembodiment, the method may comprise combining from about 1 mg/ml toabout 100 mg/ml of conjugates of a hydrogel forming agent and aflavonoid; from about 0.01 mM to about 5 mM peroxide; and from about0.01 units/ml to about 10 units/ml peroxidase; thereby producing ahydrogel that is capable of adhesion of cells and which comprisesenzymatically cross-linked conjugates of a hydrogel forming agent and aflavonoid.

As used herein a “hydrogel that is capable of adhesion of cells”, is ahydrogel which can adhere cells and from which adherent cells are notreadily dislodged upon rinsing with phosphate-buffered saline (PBS).

It will be understood that a small number of cells may adhere tohydrogels not capable of adhesion of cells, for example, the hydrogelsin the present Examples formed by only oxygenic cross-linking. However,following rising with PBS, only a minimal number of cells remain adheredto such hydrogels as compared to hydrogels that are capable of adhesionof cells.

Thus, a skilled person can readily determine if a hydrogel is a hydrogelthat is capable of adhesion of cells by contacting the hydrogel withcells and subsequently rinsing the cells with PBS and observing thenumber of remaining adherent cells.

For example, to determine if a hydrogel is capable of cell adhesion,cells may be seeded in 96-well plates coated with the hydrogel to betested. The cells may then be gently rinsed with approximately 100 μl ofthe PBS at approximately 37° C. and the number of cells that remainadhered to the hydrogel observed. A hydrogel capable of adhesion ofcells, as described herein, is a hydrogel to which a substantial numberof the cells remain adhered after rinsing with PBS.

The cells that the presently described hydrogel is capable of adheringor cell that is to be adhered to the presently described hydrogel may beany cell to which a flavonoid or biological agent, including ananti-cancer agent, is desired to be delivered or which is desired to beproliferated or adhered to the described hydrogel. In differentembodiments, the cell may be a non-cancer cell, a cancer cell, or a stemcell, as defined below. The cell may be in vitro or in vivo. In oneembodiment, the cell may be a cell located in a subject in need oftreatment for cancer. In one embodiment, the subject is human.

It will be understood that the extent of enzymatic cross-linking in thehydrogel contemplated herein, and consequently the number of cellsadhering to the hydrogel may be varied depending on the intended use ofthe hydrogel. For example, if the hydrogel is to be used to proliferatenon-cancer cells, the hydrogel should contain sufficient enzymaticcross-linking for adhesion of an appropriate number of non-cancer cellsto result in the desired cell proliferation on the hydrogel. In anotherexample, if the hydrogel is to be used in the treatment of cancer, thehydrogel should contain sufficient cross-linking for adhesion of anappropriate amount of cancer cells to effectively deliver thetherapeutic agents contained in the hydrogel to a sufficient number ofcancer cells to achieve the desired result, for example inhibition,slowing or reduction of tumour growth. Furthermore, it will beunderstood that the extent of enzymatic cross-linking required toachieve adhesion of the desired number of cells may differ depending onthe type of cells that are the cells.

Thus, in the hydrogels described herein, the extent of enzymaticcross-linking may be correlated to the number of adherent cells requiredfor the intended use of the hydrogel or the type of cell that is desiredto be adhered.

In one embodiment, the extent of enzymatic cross-linking is correlatedto the number of cells that can be effectively targeted by the amount oftherapeutic agents present in the hydrogel.

In another embodiment, there is provided a method for producing aflavonoid conjugate hydrogel without the addition of an exogenousperoxide or a peroxidase or without the addition of an exogenousperoxide. The presently described method for producing a flavonoidconjugate hydrogel without the addition of a peroxidase mayadvantageously avoid any possible immunogenicity concerns associatedwith the use of peroxidase in forming hydrogels.

Formation of Flavonoid Conjugates

As discussed, conjugation of an agent, including a hydrogel formingagent, to a flavonoid can increase the availability of flavonoidcompounds while augmenting the flavonoid's biological or pharmacologicalproperties.

The flavonoid conjugate presently described may be comprised of anysuitable hydrogel forming agent and any flavonoid, as described below.

A hydrogel forming agent for use in the methods and hydrogels describedherein may be any chemical group or moiety that can be conjugated to aflavonoid to form a molecule that is capable of being formed into ahydrogel. Thus, the hydrogel forming agent should be hydrophilic, waterinsoluble and have good swellability characteristics. Furthermore, itwill be understood that the hydrogel forming agent should be non-toxic,biocompatible and suitable for pharmacological use.

The hydrogel forming agent may also have other desirable properties, forexample, the hydrogel forming agent may have low immunogenicity, and itmay be biodegradable or non-biodegradable depending on the desiredbiological application of the composition, for example, for controlledrelease of flavonoids at a particular site in a body.

In different embodiments, the hydrogel forming agent is a protein,polysaccharide, monomer, polymer or copolymer or derivatives, polymersor copolymers thereof.

In one embodiment, the hydrogel forming agent may be a proteinincluding, for example, gelatin.

In another embodiment, the hydrogel forming agent may be apolysaccharide including, for example, dextran or chitosan.

In one embodiment, the hydrogel forming agent may be a hydrophilicmonomer including, for example, (2-hydroxyethyl) methacrylate andethyleneglycol bismethacrylate.

In another embodiment, the hydrogel forming agent may be a copolymerincluding, for example, poly(lactic-co-glycolic acid)

In particular embodiments the hydrogel forming agent is a polymerincluding, for example, agarose, poly(ethylene glycol), alginate orhyaluronic acid. In particular embodiments the polymer may be abiodegradable polymer. The polymer may be a natural or a syntheticpolymer. The polymer may be chosen to have desired swellabilitycharacteristics and to have appropriate groups available forcross-linking of the polymer moieties.

In different embodiments, the polymer may be derived from a single typeor species of monomer or may be a copolymer derived from two or moretypes or species of monomers. In one example, the polymer may becomprised of repeating units of one type of monomer. In another examplethe polymer may be comprised of alternating units of two or more typesof monomers. In still another example, the polymer may be comprised of asequence of monomers comprised of two or more types of monomers that isrepeated throughout the polymer.

In other embodiments, the polymer that is the hydrogel forming agent maybe derived from polymers that are combined to form a larger polymer thatis the hydrogel forming agent. In different embodiments, the polymerscombined to form the polymer that is the hydrogel forming agent are of asingle type or species of polymer or of two or more different types ofspecies of polymer. In one embodiment, the polymer that is the hydrogelforming agent is comprised of repeating units of one type of polymer. Inanother example, one type of polymer may be combined with another typeof polymer to form the polymer that is the hydrogel forming agent. Inyet another example, a polymer may be combined with a monomer to formthe polymer that is the hydrogel forming agent.

In a particular embodiment, the hydrogel forming agent is the polymerhyaluronic acid (HA). In different embodiments, the polymer isaldehyde-derivatized hyaluronic acid, hyaluronic acid conjugated withaminoacetylaldehyde diethylacetal, or either of the aforementionedhyaluronic acid polymers derivatized with tyramine. Methods ofsynthesizing such HA polymers are known in the art and have beendescribed for example in international application WO 2006/124000 and USapplication 2008/102052, the content of which are fully incorporatedherein.

The flavonoid may be any flavonoid from the general class of moleculesderived from a core phenylbenzyl pyrone structure, and includesflavones, isoflavones, flavonols, flavanones, flavan-3-ols, catechins,anthocyanidins and chalcones.

In particular embodiments the flavonoid is a catechin or acatechin-based flavonoid. A catechin, or a catechin-based flavonoid isany flavonoid that belongs to the class generally known as catechins (orflavan-3-ol derivatives), and includes catechin and catechinderivatives, including epicatechin, epigallocatechin, catechin,epicatechin gallate and epigallocatechin gallate, and including allpossible stereoisomers of catechins or catechin-based flavonoids. Inparticular embodiments, the catechin-based flavonoid is (+)-catechin or(−)-epigallocatechin gallate. In a particular embodiment, thecatechin-based flavonoid is epigallocatechin gallate.

A catechin-based flavonoid to be conjugated to a hydrogel forming agentmay be a single monomeric unit of a catechin-based flavonoid or it maybe an oligomer of one or more catechin-based flavonoids. As statedabove, conjugation of a hydrogel forming agent to a flavonoid can resultin augmentation of the flavonoid's biological or pharmacologicalproperties. Furthermore, oligomers of catechin-based flavonoids tend tohave amplified or augmented levels of the biological and pharmacologicalproperties associated with catechin-based flavonoids, and may even havereduced pro-oxidant effects that are sometimes associated with monomericcatechin-based flavonoids. Thus in one embodiment, an oligomerizedcatechin-based flavonoid having amplified or augmented flavonoidproperties is conjugated to the hydrogel forming agent.

Oligomers of catechin-based flavonoids that can be conjugated tohydrogel forming agents, such as polymers, are known, and includeoligomers prepared through enzyme-catalyzed oxidative coupling andthrough aldehyde-mediated oligomerization, for example as described inpublished international application WO 2006/124000 and published USapplication 2008/102052, the contents of which are fully incorporated byreference herein.

An aldehyde-mediated oligomerization process results in an unbranchedoligomer that has defined linkages, for example through carbon-carbonlinkages such as CH—CH₃ bridges linked from the C6 or C8 position on theA ring of one monomer to the C6 or C8 position on the A ring of the nextmonomer, including in either possible stereoconfiguration, whereapplicable. Thus, the CH—CH₃ linkage may be between the C6 position ofthe A ring of one monomer and either of the C6 or C8 position of thenext monomer or it may be between the C8 position of the A ring of thefirst monomer and either of the C6 or C8 position of the next monomer.

The oligomer of catechin-based flavonoid to be conjugated to thehydrogel forming agent, for example a polymer, may be of 2 or moremonomeric units linked together. In certain embodiments, thecatechin-based flavonoid oligomer has from 2 to 100 monomer units, from10 to 100, from 2 to 80, from 10 to 80, from 2 to 50, from 10 to 50,from 2 to 30, from 10 to 30, from 20 to 100, from 30 to 100 or from 50to 100 monomeric units.

The hydrogel forming agent may be conjugated to the flavonoid by anysuitable means known in the art that provides attachment of the hydrogelforming agent to the flavonoid to form a conjugate capable of beingformed into a hydrogel, while maintaining or augmenting the biologicaland pharmacological properties of the flavonoid and without disruptionof the polyphenol structure of the flavonoid.

In one embodiment, a hydrogel forming agent may be conjugated to aflavonoid by “aldehyde mediated conjugation” wherein the hydrogelforming agent is reacted with the flavonoid in the presence of an acidcatalyst, the hydrogel forming agent having a free aldehyde group, or agroup that is able to be converted to a free aldehyde group in thepresence of acid. Aldehyde-mediated conjugation of a hydrogel formingagent to a flavonoid can result in attachment of the hydrogel formingagent at the C6 and/or C8 position of the flavonoid A ring, which doesnot disrupt or affect the B and C rings of the flavonoid or the varioushydroxyl groups on the flavonoid. Formation of flavonoid conjugates byaldehyde mediated conjugation is described in published internationalapplication WO 2006/124000 and published US application 2008/102052, thecontents of which are fully incorporated by reference herein.

In a particular embodiment, the flavonoid conjugate is comprised ofpolymer conjugated to a catechin-based flavonoid and the conjugation iscarried out by aldehyde mediated conjugation as defined above. Thus, theconjugation reaction may involve conjugation of a polymer containing afree aldehyde group or a group that is able to be converted to a freealdehyde group in the presence of acid to a catechin-based flavonoid.

The polymer may be any chemical group or moiety having a free aldehydegroup prior to conjugation with the catechin-based flavonoid, or havinga group that is converted to an aldehyde group in the presence of acid,for example an acetal group, and that can be incorporated into ahydrogel. The polymer may also be any biological polymer, modified tocontain a free aldehyde group or a group that is convertible to analdehyde in the presence of acid, for example an aldehyde-modifiedprotein, peptide, polysaccharide or nucleic acid.

The free aldehyde group on the polymer allows for the conjugation of thepolymer in a controlled manner to either the C6 or the C8 position ofthe A ring, or both, of the flavonoid structure, thus preventingdisruption of the flavonoid structure, particularly the B and C rings ofthe flavonoid, and thus preserving the beneficial biological andpharmacological properties of the flavonoid. The polymer is conjugatedto the catechin-based flavonoid via a reaction of the aldehyde group ofthe polymer with the C6 and/or the C8 position of the A ring of thecatechin-based flavonoid.

The flavonoid conjugate may be synthesized using acid catalysis of acondensation of the aldehyde group of the polymer with thecatechin-based flavonoid, or using acid to convert a functional group onthe polymer to a free aldehyde prior to condensation of the aldehydegroup with the catechin-based flavonoid.

To conjugate the polymer and the catechin-based flavonoid, the polymerand the catechin-based flavonoid may be separately dissolved in asuitable solvent. The polymer with the free aldehyde is added, forexample by dropwise addition, to the solution containing thecatechin-based flavonoid, in the presence of an acid, for example at apH from about 1 to about 5, or for example at pH of about 1. Thereaction is allowed to go to completion. Following the conjugationreaction, excess unreacted catechin-based flavonoid can be removed fromthe conjugated composition, for example by dialysis or by molecularsieving.

In another embodiment, the polymer may be dissolved in deionized ordistilled water and mixed with a solution comprising the catechin-basedflavonoid dissolved in dimethyl sulfoxide (DMSO). The pH of the solutionis adjusted to about 1 by addition of an acid, for example HCl and thereaction is allowed to go to completion, for example by stirring at roomtemperature for about 24 hours. Following the conjugation reaction, theconjugate may be purified from the solution, for example by dialysis.

The ratio of flavonoid to hydrogel forming agent, may be varied, so thatthere is only one hydrogel forming agent moiety attached to theflavonoid, or so that there is a flavonoid attached at more than oneposition on the hydrogel forming agent or so that the flavonoid has twohydrogel forming agent moieties attached, for example one at either ofthe C6 and C8 positions of a catechin-based flavonoid.

The ratio of hydrogel forming agent to flavonoid in the conjugate can becontrolled through the ratio of starting reagents. For example, when themolar ratio of hydrogel forming agent to flavonoid is about 1, a singlehydrogel forming agent moiety will be attached to a single flavonoidmoiety (either monomeric or oligomeric may be used). However, at higherconcentrations of hydrogel forming agent, for example at a 10:1 molarratio of hydrogel forming agent to flavonoid, a composition having atri-block structure of hydrogel forming agent-flavonoid-hydrogel formingagent may be obtained.

Similarly, the degree of conjugation of the hydrogel forming agent withthe flavonoid can be varied by varying the concentrations of hydrogelforming agent and flavonoid in the conjugation reaction. The “degree ofconjugation” as used herein refers to the number of flavonoid moleculesper 100 units of hydrogel forming agent. For example, a 50% degree ofconjugation means that there are 50 flavonoid molecules per 100 units ofhydrogel forming agent.

In a particular example, since hyaluronic acid (HA) has multiple sitesthat may react with a flavonoid during the conjugation reaction, byvarying the concentration of the flavonoid in the starting reaction, itis possible to vary the degree of conjugation between the HA polymer andthe flavonoid.

For certain hydrogel forming agents, including HA, conjugation to aflavonoid may be accomplished by conversion of particular groups on thehydrogel forming agent to groups that are capable of conjugation with aflavonoid. For example, the conjugation of a HA polymer with acatechin-based flavonoid may be accomplished by conversion of groups onthe HA polymer to free aldehyde groups.

The ratio of hydrogel forming agent to flavonoid in the startingreagents for forming the flavonoid conjugates may be varied to adjustthe degree of conjugation of the hydrogel forming agent with theflavonoid in the resulting flavonoid conjugates and thus the ratio ofhydrogel forming agent to flavonoid present in a hydrogel formed fromthese flavonoid conjugates. Alternatively, additional hydrogel formingagent that has not been conjugated can be added to the solution forforming a hydrogel prior to cross-linking of the hydrogel so that someof the hydrogel forming agent molecules in the hydrogel will not beconjugated to the flavonoid.

In one embodiment, the flavonoid conjugates used in forming theflavonoid hydrogel have a degree of conjugation of from about 1% toabout 90%. In another embodiment, the flavonoid conjugates used informing the flavonoid hydrogel have a degree of conjugation o of fromabout 1% to about 50%. In another embodiment, the flavonoid conjugatesused in forming the flavonoid hydrogel have a degree of conjugation offrom about 2% to about 10%.

In a particular embodiment, the flavonoid conjugate is a conjugate of HAand EGCG and the conjugate is synthesized in a two-step procedure. Inthe first step protected aldehyde groups are introduced to HA byconjugating diethoxyethyl amine (DA) to HA though NHS/EDC chemistry. Theresulting conjugates, HA-DA, generally have a substitution degree(number of carboxyl groups converted to DA in every 100 disaccharideunit) of 10%. HA-DA is then deprotected at a pH of 1 to allowconjugation of EGCG to the aldehyde groups. The conjugation degree ofEGCG may be between 1 to 2.5% in the dimer conformation. The resultingHA-EGCG conjugate is soluble in water and can form hydrogels via thecrosslinking of the EGCG moieties.

Formation of the Flavonoid Conjugate Hydrogel

There is presently provided a method for forming a hydrogel capable ofadhesion of cells comprising enzymatically cross-linked flavonoidconjugates (referred to herein as an “enzymatically cross-linkedflavonoid conjugate hydrogel”). The flavonoid conjugates may be aconjugate of any suitable hydrogel forming agent and any suitableflavonoid as describe herein. In one embodiment, the flavonoid conjugateis a conjugate of a polymer and a catechin-based flavonoid. In aparticular embodiment, the flavonoid conjugate is a conjugate ofhyaluronic acid and epigallocatechin gallate.

To form the enzymatically cross-linked flavonoid conjugate hydrogeldescribed herein, the flavonoid conjugates are cross-linked using asufficient amount of peroxidase enzyme, and peroxide to form a hydrogelwith sufficient enzymatic cross-linking for adhesion of cells to thehydrogel.

Thus, the method comprises combining (i) conjugates of a hydrogelforming agent and a flavonoid; (ii) a peroxide; and (iii) a peroxidaseall at concentrations that provide sufficient enzymatic cross-linkingbetween the conjugates for adhesion of cells to the hydrogel. It will bereadily understood that the ratio of hydrogel forming agent to flavonoidin the hydrogel and the degree of conjugation of the hydrogel formingagent with the flavonoid may affect the concentrations of flavonoidconjugates, peroxide and peroxidase required to form a hydrogel withsufficient enzymatic cross-linking of the flavonoid conjugates foradhesion of cells.

As discussed above, enzymatic cross-linking of the flavonoid conjugatesis formed by oxidation catalysed by a peroxidase enzyme. The peroxidaseused may be any peroxidase enzyme that can catalyse reduction ofperoxide, and thus result in concomitant oxidation and thuscross-linking between two phenolic hydroxyl groups on flavonoid moietieswithin the conjugates when used in the present methods. In differentembodiments, the peroxidase may be horseradish peroxidase, pegylatedhorse radish peroxidase or laccase. Conveniently, the peroxidase enzymemay be horse radish peroxidase (HRP), which may be readily purchased.

The peroxidase enzyme is mixed with the flavonoid conjugates togetherwith a peroxide in order to effect enzymatic cross-linking of theconjugates to form the enzymatically cross-linked flavonoid conjugatehydrogel as described herein.

The peroxide may be any form of peroxide that can act as a substrate forthe peroxidase enzyme to activate the cross-linking activity of theperoxidase enzyme. In particular embodiments, the peroxide used ishydrogen peroxide, H₂O₂.

The concentration of peroxidase enzyme used in the synthesis of aflavonoid conjugate hydrogel can affect the amount of enzymaticcross-linking present in the hydrogel. Thus, the peroxidase enzyme isprovided at a concentration that will result in sufficient amount ofenzymatic cross-linking in the flavonoid conjugate hydrogel to achieveadhesion of cells.

It will be understood by a person skilled in the art that theconcentration of peroxidase enzyme required to form a sufficient amountof enzymatic cross-linking in a flavonoid conjugate hydrogel foradhesion of cells will vary depending on a number of factors includingthe type and concentration of the hydrogel forming agent, the type andconcentration of the flavonoid, the type and concentration of flavonoidconjugates, the ratio of hydrogel forming agent to flavonoid in thehydrogel, the degree of conjugation of the hydrogel forming agent withthe flavonoid, the concentration of peroxide, the temperature at whichthe hydrogel is synthesized or the pH at which the hydrogel issynthesized.

For example, the above factors may affect the rate of enzymaticcross-linking and thus the amount of peroxidase enzyme required to forma sufficient amount of enzymatic cross-linking in the flavonoidconjugate hydrogel for adhesion of cells.

The present inventors have discovered that a flavonoid conjugatehydrogel comprising a sufficient amount of enzymatic cross-linking foradhesion of cells may be formed by mixing: from about 0.1 mg/ml to about500 mg/ml of conjugates of a hydrogel forming agent and a flavonoid,from about 0.001 mM to about 50 mM peroxide; and from about 0.001units/ml to about 10 units/ml peroxidase.

Thus there is presently provided, a method for producing a hydrogel thatis capable of adhesion of cells and which comprises enzymaticallycross-linked conjugates of a hydrogel forming agent and a flavonoid, themethod comprising combining from about 0.1 mg/ml to about 500 mg/ml ofconjugates of the hydrogel forming agent and the flavonoid from about0.001 mM to about 50 mM peroxide; and from about 0.001 units/ml to about10 units/ml peroxidase; thereby producing the hydrogel.

In different embodiments, the concentration of flavonoid conjugates maybe from about 0.1 mg/ml to about 500 mg/ml, from about 1 mg/ml to about100 mg/ml, at least about 0.1 mg/ml, at least about 0.3 mg/ml, at leastabout 0.5 mg/ml, at least about 0.7 mg/ml, at least about 1 mg/ml, atleast about 5 mg/ml, at least about 10 mg/ml, at least about 15 mg/ml,at least about 17 mg/ml, at least about 17.5 mg/ml, at least about 20mg/ml, at least about 25 mg/ml, at least about 30 mg/ml, at least about35 mg/ml, at least about 40 mg/ml, at least about 45 mg/ml, at leastabout 50 mg/ml, at least about 55 mg/ml, at least about 60 mg/ml, atleast about 65 mg/ml, at least about 70 mg/ml, at least about 75 mg/ml,at least about 80 mg/ml, at least about 85 mg/ml, at least about 90mg/ml, at least about 95 mg/ml, at least about 100 mg/ml, at least about200 mg/ml, at least about 250, at least about 300 mg/ml, at least about350 mg/ml, at least about 400 mg/ml, at least about 450 mg/ml, at leastabout 500 mg/ml; the concentration of peroxide may be from about 0.001mM to about 50 mM, from about 0.01 mM to about 5 mM, at least about0.001 mM, at least about 0.003 mM, at least about 0.005 mM, at leastabout 0.007 mM, at least about 0.01 mM, at least about 0.014 mM, atleast about 0.02 mM, at least about 0.03 mM, at least about 0.04 mM, atleast about 0.05 mM, at least about 0.06 mM, at least about 0.07 mM, atleast about 0.08 mM, at least about 0.09 mM, at least about 0.1 mM, atleast about 0.2 mM, at least about 0.3 mM, at least about 0.4 mM, atleast about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, atleast about 0.8 mM, at least about 0.9 mM, at least about 1 mM, at leastabout 1.5 mM, at least about 2 mM, at least about 2.5 mM, at least about3 mM, at least about 3.5 mM, at least about 4 mM, at least about 4.5 mM,at least about 5 mM, at least about 10 mM, at least about 15 mM, atleast about 20 mM, at least about 25 mM, at least about, 30 mM, at leastabout 35 mM, at least about 40 mM, at least about 45 mM, at least about50 mM; and the concentration of peroxidase may be from about 0.001units/ml to about 10 units/ml, from about 0.01 units/ml to about 10units/ml, at least about 0.001 units/ml, at least about 0.003 units/ml,at least about 0.005 units/ml, at least about 0.007 units/ml, at leastabout 0.01 units/ml, at least about 0.02 units/ml, at least about 0.03units/ml, at least about 0.04 units/ml, at least about 0.05 units/ml, atleast about 0.06 units/ml, at least about 0.07 units/ml at least about0.08 units/ml, at least about 0.09 units/ml, at least about 0.1units/ml, at least about 0.5 units/ml, at least about 1.0 units/ml, atleast about 1.5 units/ml, at least about 2 units/ml, at least about 2.3units/ml, at least about 2.5 units/ml, at least about 3.0 units/ml, atleast about 3.2 units/ml, at least about 3.5 units/ml, at least about4.0 units/ml, at least about 4.1 units/ml, at least about 4.5 units/ml,at least about 4.9 units/ml, at least about 5.0 units/ml, at least about5.5 units/ml, at least about 6.0 units/ml, at least about 6.5 units/ml,at least about 7.0 units/ml, at least about 7.5 units/ml, at least about8.0 units/ml, at least about 8.5 units/ml, at least about 9.0 units/ml,at least about 9.5 units/ml, or at least about 10 units/ml. Inalternative embodiments, there is provided any combination of theconcentrations of flavonoid conjugates, concentrations of peroxide andconcentrations of peroxidase provided herein.

Alternatively, the concentration of flavonoid conjugates may be fromabout 0.1 mg/ml to about 500 mg/ml, from about 1 mg/ml to about 100mg/ml, at least about 0.1 mg/ml, at least about 0.3 mg/ml, at leastabout 0.5 mg/ml, at least about 0.7 mg/ml, at least about 1 mg/ml, atleast about 5 mg/ml, at least about 10 mg/ml, at least about 15 mg/ml,at least about 17 mg/ml, at least about 17.5 mg/ml, at least about 20mg/ml, at least about 25 mg/ml, at least about 30 mg/ml, at least about35 mg/ml, at least about 40 mg/ml, at least about 45 mg/ml, at leastabout 50 mg/ml, at least about 55 mg/ml, at least about 60 mg/ml, atleast about 65 mg/ml, at least about 70 mg/ml, at least about 75 mg/ml,at least about 80 mg/ml, at least about 85 mg/ml, at least about 90mg/ml, at least about 95 mg/ml, at least about 100 mg/ml, at least about200 mg/ml, at least about 250, at least about 300 mg/ml, at least about350 mg/ml, at least about 400 mg/ml, at least about 450 mg/ml, at leastabout 500 mg/ml. Alternatively, the concentration of peroxide may befrom about 0.001 mM to about 50 mM, from about 0.01 mM to about 5 mM, atleast about 0.001 mM, at least about 0.003 mM, at least about 0.005 mM,at least about 0.007 mM, at least about 0.01 mM, at least about 0.014mM, at least about 0.02 mM, at least about 0.03 mM, at least about 0.04mM, at least about 0.05 mM, at least about 0.06 mM, at least about 0.07mM, at least about 0.08 mM, at least about 0.09 mM, at least about 0.1mM, at least about 0.2 mM, at least about 0.3 mM, at least about 0.4 mM,at least about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, atleast about 0.8 mM, at least about 0.9 mM, at least about 1 mM, at leastabout 1.5 mM, at least about 2 mM, at least about 2.5 mM, at least about3 mM, at least about 3.5 mM, at least about 4 mM, at least about 4.5 mM,at least about 5 mM, at least about 10 mM, at least about 15 mM, atleast about 20 mM, at least about 25 mM, at least about, 30 mM, at leastabout 35 mM, at least about 40 mM, at least about 45 mM, at least about50 mM. Alternatively, the concentration of peroxidase may be from about0.001 units/ml to about 10 units/ml, from about 0.01 units/ml to about10 units/ml, at least about 0.001 units/ml, at least about 0.003units/ml, at least about 0.005 units/ml, at least about 0.007 units/ml,at least about 0.01 units/ml, at least about 0.02 units/ml, at leastabout 0.03 units/ml, at least about 0.04 units/ml, at least about 0.05units/ml, at least about 0.06 units/ml, at least about 0.07 units/ml atleast about 0.08 units/ml, at least about 0.09 units/ml, at least about0.1 units/ml, at least about 0.5 units/ml, at least about 1.0 units/ml,at least about 1.5 units/ml, at least about 2 units/ml, at least about2.3 units/ml, at least about 2.5 units/ml, at least about 3.0 units/ml,at least about 3.2 units/ml, at least about 3.5 units/ml, at least about4.0 units/ml, at least about 4.1 units/ml, at least about 4.5 units/ml,at least about 4.9 units/ml, at least about 5.0 units/ml, at least about5.5 units/ml, at least about 6.0 units/ml, at least about 6.5 units/ml,at least about 7.0 units/ml, at least about 7.5 units/ml, at least about8.0 units/ml, at least about 8.5 units/ml, at least about 9.0 units/ml,at least about 9.5 units/ml, or at least about 10 units/ml.

In one embodiment, the concentration of the flavonoid conjugates is fromabout 1 mg/ml to about 100 mg/ml, the concentration of the peroxide isfrom about 0.01 mM to about 5 mM and the concentration of the peroxidaseis from about 0.01 units/ml to about 10 units/ml.

There is also presently provided a hydrogel capable of adhesion of cellsand which comprises enzymatically cross-linked conjugates of a hydrogelforming agent and a flavonoid, the hydrogel produced by a methodcomprising combining from about 0.1 mg/ml to about 500 mg/ml ofconjugates of the hydrogel forming agent and the flavonoid; from about0.001 mM to about 50 mM peroxide; and from about 0.001 units/ml to about10 units/ml peroxidase.

In one embodiment, the hydrogel is formed by combining from about 1mg/ml to about 100 mg/ml of conjugates of a hydrogel forming agent and aflavonoid; from about 0.01 mM to about 5 mM peroxide; and from about0.01 units/ml to about 10 units/ml peroxidase.

It will be understood that the amount of enzymatic cross-linking thatwill be sufficient for adhesion of cells may vary depending on the typeof cell to be adhered. For example, adhesion of non-cancer cells to thehydrogel may require a different amount of enzymatic cross-linking thanadhesion of cancer cells. In addition, the incorporation of bioactiveagents may affect the amount of enzymatic cross-linking that will berequired for adhesion of cells.

It will be understood that the amount of enzymatic cross-linking may bemodified to provide adhesion of an appropriate number of cells to thehydrogel to achieve a desired result. For example, the enzymaticallycross-linked flavonoid conjugate hydrogel described herein may beproduced such that it comprises a sufficient amount of enzymaticcross-linking to adhere an appropriate number of cells to result in cellproliferation on the hydrogel. In another example, the enzymaticallycross-linked flavonoid conjugate hydrogel described herein may beproduced such that it comprises a sufficient amount of enzymaticcross-linking to adhere an appropriate amount of cells to the hydrogelthat can be treated by the amount of flavonoid and any other activeagent present in the hydrogel. In another embodiment, the enzymaticallycross-linked flavonoid conjugate hydrogel described herein may beproduced such that it comprises a sufficient amount of enzymaticcross-linking to adhere an appropriate amount of cells to the hydrogelfor treatment of a disease or disorder by the flavonoids or active agentpresent in the hydrogel. In a particular embodiment, the enzymaticallycross-linked flavonoid conjugate hydrogel described herein may beproduced such that it comprises a sufficient amount of enzymaticcross-linking to adhere an appropriate amount of cancer cells to thehydrogel for the treatment of cancer.

A person skilled in the art can use known methods and techniques todetermine, based on the above factors, the relative concentration offlavonoid conjugates, peroxidase enzyme, and peroxide required to form asufficient amount of enzymatic cross-linking for adhesion of cells orfor adhesion of an appropriate number of cells for a desired result. Forinstance, whether a sufficient amount of enzymatic cross-linking iscontained within a specific hydrogel preparation for adhesion of cellscan readily be determined by culturing cells in the presence of thehydrogel, for example as described in the following Examples, and usingtechniques known in the art to determine or monitor cell adhesion,proliferation or viability. Furthermore, a skilled person would be ableto determine the appropriate conditions, such as concentration ofperoxide and concentration of flavonoid conjugates, required tosynthesize a hydrogel.

In a particular embodiment, the flavonoid conjugates are hyaluronicacid-epigallocatechin gallate (HA-EGCG) conjugates, the peroxidase ishorseradish peroxidase and the peroxide is hydrogen peroxide. HA, amajor component of the extracellular matrix, is a desirable backbonepolymer for hydrogels due to its high biocompatability andbiodegradability [15]. Furthermore, HA is a chemoattractant and maydirect cells, such as cancer cell, to migrate towards the hydrogel [14].EGCG can bind proteins, and thus an HA-EGCG hydrogel can provide theadvantage of protein-EGCG interaction [12] for immobilizing proteins,such as bioactive agents, in the gel, which can be combined with thetherapeutic benefits of EGCG.

Thus in one embodiment, a hyaluronic acid-epigallocatechin gallate(HA-EGCG) hydrogel is formed by mixing HA-EGCG conjugates withhorseradish peroxidase in the presence of hydrogen peroxide. In aparticular embodiment, the concentration of the HA-EGCG conjugates isfrom about 0.1 mg/ml to about 500 mg/ml, the concentration of hydrogenperoxide is from about 0.001 mM to about 50 mM; and the concentration ofhorseradish peroxidase is from about 0.001 units/ml to about 10 units/mlperoxidase. In another embodiment, the concentration of the HA-EGCGconjugates is at least about 17.5 mg/ml, the concentration of thehydrogen peroxide is at least about 0.014 mM, and the concentration ofthe horseradish peroxidase is at least about 2.3 units/ml.

The flavonoid conjugates may be combined with the peroxidase andperoxide using any suitable method known in the art. For example, asolution containing the flavonoid conjugates may be prepared or obtainedfirst. The flavonoid conjugate solution may be prepared in any suitablemanner, for example by providing the flavonoid conjugate solution inDulbecco's Modified Eagle's Medium containing 10% fetal bovine serum.The peroxidase and peroxide may then be added to the flavonoid conjugatesolution.

Subsequent to the addition of the peroxidase and peroxide, the solutionmay be quickly poured into a mold to form a desired shape before thecross-linking reaction is completed. For example, the hydrogel may beformed into a slab suitable for application as a wound dressing.Alternatively, the hydrogel may be formed within the same vessel inwhich the peroxidase, peroxide and flavonoid conjugate solution arecombined. For example, the peroxidase, peroxide and the flavonoidconjugate solution may be combined in a cell culture plate and thehydrogel formed within that culture plate.

The components of the hydrogel may also be injected and reacted to formthe hydrogel in vivo, for example in a living tissue, organism or livingbody including a human living body. Hydrogels may be formed in vivo byinjecting the uncross-linked flavonoid conjugates together with thecross-linking enzyme and enzyme activator, or injecting the mixture ofthe components prior to completion of the cross-linking reaction. Such ahydrogel is useful for drug delivery to a specific site in a body, orfor tissue engineering.

In another embodiment, there is provided a method for producing ahydrogel comprising conjugates of a hydrogel forming agent and aflavonoid, the method comprising combining the conjugates in a solutionin the absence of an exogenously added peroxide and in the absence of aperoxidase.

The present inventors have discovered that flavonoid conjugate hydrogelscan be formed through air auto-oxidation without the addition ofperoxide or peroxidase. Forming the hydrogels through airauto-oxidation, without the use of peroxidase, may advantageously avoidpossible immunogenicity concerns associated with some peroxidases, suchas horseradish peroxidase.

Furthermore, the gelation rate and stiffness of the flavonoid conjugatehydrogels formed by this method [herein referred to as “auto-oxidationflavonoid conjugate hydrogel”] may be controlled by modifying the pH ofthe solution comprising the flavonoid conjugates. EGCG undergoesoxidation in the presence of di-oxygen molecules to form EGCG quinoneand hydrogen peroxide (H₂O₂) [52-55] and the EGCG quinone can react withEGCG to form EGCG dimer. The rate of oxidation has been shown toincrease with pH [52]. The present inventors have discovered that thegelation rate of the auto-oxidation flavonoid conjugate hydrogeldescribed herein may be reduced by increasing the pH of the solutioncomprising the flavonoid conjugates. In particular embodiments, thegelation rate of the auto-oxidation flavonoid conjugate hydrogeldescribed herein may be controlled by modifying the pH of the solutioncomprising the flavonoid conjugates between from about 3 to about 10. Inone embodiment, the gelation rate of the auto-oxidation flavonoidconjugate hydrogel described herein may be controlled by modifying thepH of the solution between from about 6 to about 8. A skilled personwould be able to adjust the pH of the hydrogel, within the rangesdescribed herein, to achieve a desired gelation rate.

In a particular embodiment, the method for producing the auto-oxidationflavonoid conjugate hydrogel described herein without the addition of anexogenous peroxide or a peroxidase may further comprise addition ofcatalase to the solution comprising the flavonoid conjugates. Catalaseis an enzyme that catalyzes the decomposition of hydrogen peroxide towater and oxygen. In particular embodiments, catalase may be added tothe solution comprising the flavonoid conjugates to remove the H₂O₂generated during air-autoxidation of the flavonoid conjugates and reducethe gelation time of the flavonoid conjugate hydrogel.

In another embodiment, there is provided a method for producing ahydrogel comprising conjugates of a hydrogel forming agent and aflavonoid, the method comprising combining the conjugates and aperoxidase in a solution in the absence of an exogenously addedperoxide.

Peroxidases, including, for example, horseradish peroxidase, are able tocatalyze a variety of substrates, including flavonoids, including EGCG,through reaction with H₂O₂. This reaction has been explored to formhydrogels rapidly by using tyramine as the crosslinking moiety [56].Typically, exogenous H₂O₂ needs to added to initiate the enzymaticreaction, as in the case of hyaluronic acid-tyramine hydrogel system.The present inventors have shown that H₂O₂ generated as a result ofair-autoxidation of flavonoid conjugates can be used for theperoxidase-mediated crosslinking reactions, thus eliminating the needfor exogenously added H₂O₂.

In different embodiments of the methods of forming an auto-oxidationflavonoid conjugate hydrogel described herein the concentration offlavonoid conjugates in the solution may be from about 0.1 mg/ml toabout 500 mg/ml, from about 1 mg/ml to about 100 mg/ml, at least about0.1 mg/ml, at least about 0.3 mg/ml, at least about 0.5 mg/ml, at leastabout 0.7 mg/ml, at least about 1 mg/ml, at least about 5 mg/ml, atleast about 10 mg/ml, at least about 15 mg/ml, at least about 17 mg/ml,at least about 17.5 mg/ml, at least about 20 mg/ml, at least about 25mg/ml, at least about 30 mg/ml, at least about 35 mg/ml, at least about40 mg/ml, at least about 45 mg/ml, at least about 50 mg/ml, at leastabout 55 mg/ml, at least about 60 mg/ml, at least about 65 mg/ml, atleast about 70 mg/ml, at least about 75 mg/ml, at least about 80 mg/ml,at least about 85 mg/ml, at least about 90 mg/ml, at least about 95mg/ml, at least about 100 mg/ml, at least about 200 mg/ml, at leastabout 250, at least about 300 mg/ml, at least about 350 mg/ml, at leastabout 400 mg/ml, at least about 450 mg/ml, at least about 500 mg/ml.

There is also provided a hydrogel comprising conjugates of a hydrogelforming agent and a flavonoid, the hydrogel produced by the methoddescribed herein for producing a hydrogel comprising conjugates of ahydrogel forming agent and a flavonoid, the method comprising combiningthe conjugates in a solution in the absence of an exogenously addedperoxide and in the absence of a peroxidase.

Also provided is a hydrogel comprising conjugates of a hydrogel formingagent and a flavonoid, the hydrogel produced by the method describedherein for producing a hydrogel comprising conjugates of a hydrogelforming agent and a flavonoid, the method comprising combining theconjugates with a peroxidase in a solution in the absence of anexogenously added peroxide.

Optionally, a bioactive agent may be incorporated in the flavonoidconjugate hydrogels described herein, including by mixing in thesolution prior to cross-linking or by addition after formation of thehydrogel.

The bioactive agent may be any agent that has a biological,pharmacological or therapeutic effect in a body, and includes withoutlimitation a protein, a nucleic acid, a small molecule or a drug. Abioactive agent that is a protein may be for example a peptide, anantibody, a hormone, an enzyme, a growth factor, or a cytokine. Abioactive agent that is a nucleic acid may be for example singlestranded or double stranded DNA or RNA, a short hairpin RNA, an siRNA,or may comprise a gene encoding a therapeutic product. Also included inthe scope of bioactive agent are antibiotics, chemotherapeutic agents,antihypertensive agents, anti-cancer agents, anti-bacterial agents,anti-neoplastic agents, anti-thrombotic agents, vasodilatory agents,anti-oxidants, anti-mutagenic agents, anti-carcinogenic agents,anti-hypercholesterolemic agents, anti-viral agents andanti-inflammatory agents.

The bioactive agent may be added to the hydrogel solution beforegelation of the hydrogel or may be injected along with the othercomponents of the hydrogel such that the bioactive agent is incorporatedin the hydrogel when the hydrogel forms in vivo. The bioactive agent maybe included in the flavonoid conjugate hydrogel to be simultaneouslydelivered to a cell or to a target site in the body.

If an anti-cancer agent is included in the flavonoid conjugate gel,therapeutic synergism may be provided by the combination of the EGCGwhen delivered in combination with anti-cancer agents [13]. Thus, in oneembodiment, the bioactive agent is an anti-cancer agent. As used herein,“anti-cancer agent” refers to any agent that has an anti-cancer effecton a cell, including an anti-tumour effect, such as a cytotoxic,apoptotic, anti-mitotic anti-angiogenesis or inhibition of metastasiseffect. The anti-cancer effect is intended to include inhibition orreduction of tumour cell growth, inhibition or reduction ofcarcinogenesis, killing of tumour cells, or inhibition or reduction ofcarcinogenic or tumourogenic properties of a cell, including a tumourcell. The anti-cancer agent may be, for example, herceptin, TNP470,trastuzumab, bevacizumab, rituximab, erlotinib, daunorubicin,doxorubicin, etoposide, vinblastine, vincristine, pacitaxel,methotrexate, 5-fluorouracil, gemcitabine, arabinosylcytosine,altretamine, asparaginase, bleomycin, capecitabine, carboplatin,carmustine, BCNU, cladribine, cisplatin, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, actinomycin D, docetaxel, doxorubicin,doxorubicin, imatinib, doxorubicin liposomal, VP-16, fludarabine,gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, CPT-11,methotrexate, mitomycin, mitotane, mitoxantrone, topotecan, vinblastine,vincristine or vinorelbine or an antibody for use in immunotherapy.

The combination of the presently provided flavonoid conjugate hydrogelwith a bioactive anti-cancer agent may have a synergistic anti-cancereffect greater than the combined effects of each of the flavonoidconjugate hydrogel and the bioactive anti-cancer agent when used alone.In one embodiment, the anti-cancer agent herceptin may be incorporatedinto the presently provided flavonoid conjugate hydrogel to provide asynergistic anti-cancer effect.

In particular embodiments, the present methods for forming a flavonoidconjugate hydrogel may comprise more than one type of flavonoidconjugate. Thus, in different embodiments, the hydrogel formed by thepresent methods may comprise a mixture of two or more types of flavonoidconjugates wherein each type of flavonoid conjugate comprises adifferent combination of a hydrogel forming agent and a flavonoid. Forexample, in one embodiment, the hydrogel may comprise a mixture ofdifferent types of flavonoid conjugates wherein each type of flavonoidconjugate comprises a different hydrogel forming agent. In anotherembodiment, the hydrogel may comprise a mixture of different types offlavonoid conjugates wherein each type of flavonoid conjugate comprisesa different flavonoid. In yet another embodiment, the hydrogel maycomprise a mixture of different types of flavonoid conjugates whereineach type of flavonoid conjugate comprises a different hydrogel formingagent and a different suitable flavonoid. In a particular embodiment,the hydrogel may comprise a mixture of HA-EGCG conjugates and adifferent type of flavonoid conjugate.

The mechanical strength of the presently described flavonoid conjugatehydrogels can be modified by varying the concentration of the flavonoidconjugates and the pH of the hydrogel. In one embodiment, the mechanicalstrength of the presently described hydrogel may be modified by varyingthe concentration of the flavonoid conjugates between from about 0.1 wt% to about 20 wt % and varying the pH of the hydrogel between from about3 to about 10. In a particular embodiment, the mechanical strength ofthe presently described hydrogel may be modified by varying the the pHof the hydrogel between from about 6 to about 8. A skilled person wouldbe readily able to adjust the concentration of the flavonoid conjugateand the pH of the hydrogel, within the ranges described herein, toachieve a desired mechanical strength, for example, a particularmechanical strength to suit a particular application for the hydrogel.

Methods of Use of the Hydrogel

The enzymatically cross-linked flavonoid conjugate hydrogel describedherein can is capable of adhesion of cells. Thus, there is presentlyprovided a method for adhering a cell to a hydrogel, the methodcomprising contacting the cell with the enzymatically cross-linkedflavonoid conjugate hydrogel described herein.

Use of the presently described enzymatically cross-linked flavonoidconjugate hydrogel to adhere a cell is also provided.

The enzymatically cross-linked flavonoid conjugate hydrogel may beselected to provide a desired effect on the adhered cells. For example,the concentrations of the components used to make the hydrogel mayconveniently be selected to provide inhibition of proliferation of acancer cell. As discussed above, cell adhesion to the enzymaticallycross-linked flavonoid conjugate hydrogel described herein may provideimproved delivery of flavonoids to a cell. In addition to delivery offlavonoids to a cell by sustained release of the flavonoid into a targetsite where cells are generally located, the presently describedenzymatically cross-linked flavonoid conjugate hydrogel may facilitatedelivery of flavonoids and other active agents in the hydrogel to a cellby adhering or “trapping” cells. That is, as a result of cell adhesion,the presently described enzymatically cross-linked flavonoid conjugatehydrogels may retain cells on the hydrogel and thus in proximity to thetherapeutic agents contained in the hydrogel. Thus, the presentenzymatically cross-linked flavonoid conjugate hydrogels may provideadditional and potentially more efficient delivery of flavonoids andother active agents to cells than hydrogels to which cells do notadhere. In addition, the enzymatically cross-linked flavonoid conjugatehydrogel presently described may provide anti-metastatic effects.

FIG. 1 provides a schematic diagram of the potential biological activityof the enzymatically cross-linked flavonoid conjugate hydrogel presentlydescribed.

Thus, in one embodiment, the cell adhered to the enzymaticallycross-linked flavonoid conjugate hydrogel is a cancer cell andproliferation of the cancer cells in inhibited.

In particular embodiments, the cell may be a cell located in a subjectin need of treatment for cancer. For example, the cell may be a cellwithin a subject having cancer, a subject requiring treatment for canceror a subject in which prevention of cancer is desired. In someembodiments, the subject is a human subject.

In another embodiment, the flavonoid conjugate hydrogel may be selectedto have a sufficient amount of enzymatic cross-linking to allow forproliferation of a non-cancer cell. Thus, in one embodiment, the celladhered to the enzymatically cross-linked flavonoid conjugate hydrogelis a non-cancer cell and the non-cancer cell is proliferated.

As used herein, “cancer cell” refers to a cell that exhibits abnormalcell growth, reduced or loss of control over cell division and thepotential to invade nearby tissues. Some cancer cells may displaymetastasis in which the cell spreads to other locations in the body.Some cancer cells may form tumours. Cancer cells may include, forexample, sarcoma, carcinoma, lymphoma or blastoma cells.

The term “non-cancer cell” as used herein refers to a cell that is not acancer cell. A non-cancer cell is a cell that does not exhibit reducedor loss of control of cell division and the potential to invade nearbytissues. A non-cancer cell may include for example, a cell with normalcell growth and function, a cell with normal cell growth but abnormalcell function or a cell with abnormal cell growth that is not related toreduced or loss of control of cell division, for example a cell withreduced cell growth or abnormal cell morphology.

As used herein, the term “proliferation” and “proliferating” refers tothe growth and division of a cell.

As used herein, “inhibiting proliferation” or “inhibition ofproliferation” refers to a temporary or permanent decrease, slowing,inhibition, or termination in the proliferation of cells. For example,inhibiting proliferation may refer to suppressing cell growth and celldivision by reducing, inhibiting or modifying cell development andfunction. Inhibiting proliferation may induce cell senescence or celldeath, for example through inducing apoptosis.

As used herein, “selective anti-proliferative effect” refers to aninhibition of proliferation that affects cancer cells but does notaffect non-cancer cells or affects non-cancer cells to a lesser degree.

The presently described enzymatically cross-linked flavonoid conjugatehydrogel may induce a selective anti-proliferative effect in cancercells that adhere to the hydrogel while permitting the proliferation ofadherent non-cancer cells adhered to the hydrogel. For example, anenzymatically cross-linked HA-EGCG hydrogel prepared as described hereindid not induce any significant cytotoxicity against attached non-cancercells, but inhibited cell proliferation and induced apoptosis ofattached cancer cells. Thus, in one embodiment, the flavonoid conjugatehydrogel may be selected to have a sufficient amount of enzyme-catalyzedcross-linking to allow for proliferation of a non-cancer cell while atthe same time having a selective anti-proliferative effect in a cancercell that may be present in the same cell population as the non-cancercell, including within a subject in need of cancer treatment.

In another embodiment, there is presently provided a method fordelivering a flavonoid to a cell, the method comprising contacting theauto-oxidation flavonoid conjugate hydrogel described herein with thecell.

As used herein, “delivering” the flavonoid to a cell refers to providingthe flavonoid in sufficiently close proximity to the cell such that theflavonoid can exert its therapeutic effects on the cell. In vitro, forexample, the flavonoid may be delivered to the cell by adding thehydrogel to the cell culture media or using the hydrogel as a supportfor cell attachment and growth.

In particular embodiments, the method of adhering a cell to a hydrogel,described herein, or the method for delivering a flavonoid to a cell,described herein, may comprise administering to a subject flavonoidconjugate hydrogels comprising an effective amount of the conjugates forthe treatment of disease or disorder, including, for example, cancer.

In vivo, the flavonoid conjugate hydrogels presently provided may beadministered to a subject by any suitable manner of administration knownin the art. For example, the hydrogel may be administered by topicalapplication or by surgical insertion, including at a wound site or at asite for cancer treatment. In one embodiment, the components of thehydrogel, including a mixture of the components, may be administered byinjection at the desired target site where the components will react toform the hydrogel in vivo.

The term “effective amount” as used herein means an amount effective atdosages and for periods of time necessary to achieve a desired result.For example, the flavonoid conjugates may be administered in quantitiesand dosages necessary to deliver a flavonoid which may function toalleviate, improve, mitigate, ameliorate, stabilize, prevent the spreadof, slow or delay the progression of or cure a disease or disorder, orto inhibit, reduce or impair the activity of a disease-related enzyme. Adisease-related enzyme is an enzyme involved in a metabolic orbiochemical pathway, which when the pathway is interrupted, or whenregulatory control of the enzyme or pathway is interrupted or inhibited,the activity of the enzyme is involved in the onset or progression of adisease or disorder, for example, cancer. In another example, theflavonoid conjugates may be administered in quantities and dosagesnecessary for inducing a selective anti-proliferative effect in a cancercell adhered to the hydrogel while permitting proliferation ofnon-cancer cells on the hydrogel. In another example, the flavonoidconjugates may be administered in quantities and dosages necessary forexerting an anti-metastatic effect on a tumour. In yet another example,the flavonoid conjugates may be administered in quantities and dosagesnecessary for the treatment of cancer.

“Cancer” as used herein encompasses a class of diseases in which cellsexhibit abnormal cell growth and the potential to invade nearby tissues.In some forms of cancer, the abnormal cells may also spread to otherlocations in the body. Different types of cancer include for example,breast cancer, colorectal cancer, brain cancer, prostate cancer,cervical cancer, ovarian cancer, bone cancer, skin cancer, lung cancer,pancreatic cancer, bladder cancer, gallbladder cancer, kidney cancer,esophageal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, laryngealcancer, leukemia, multiple myeloma, oral cancer, pleural mesothelioma,small intestine cancer, testicular cancer, uterine cancer, thyroidcancer and stomach cancer.

The term “treatment” refers to an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disorder or disease, stabilization of the state of disease,prevention of development of disorder or disease, prevention of spreadof disorder or disease, delay or slowing of disorder or diseaseprogression, delay or slowing of disorder or disease onset, ameliorationor palliation of the disorder or disease state, and remission, whetherpartial or total. “Treatment” can also mean prolonging survival of asubject beyond that expected in the absence of treatment. “Treatment”can also mean inhibiting the progression of disorder or disease, slowingthe progression of disorder or disease temporarily, although in someinstances, it involves halting the progression of the disorder ordisease permanently.

The effective amount of flavonoid conjugates to be administered to asubject can vary depending on many factors such as the pharmacodynamicproperties of the flavonoid conjugates or the hydrogel comprising theflavonoid conjugates, including any bioactive agent incorporated in thehydrogel, the mode of administration, the age, health and weight of thesubject, the nature and extent of the disorder or disease state, thefrequency of the treatment and the type of concurrent treatment, if any,and the concentration and form of the hydrogel.

Furthermore, the effective amount may vary depending on the degree ofenzymatic cross-linking of the flavonoid conjugates. For example,varying the degree of enzymatic cross-linking in the enzymaticallycross-linked flavonoid conjugate hydrogel described herein may result indifferences in the amount or strength of cell adhesion and thus mayaffect the efficiency of delivery of therapeutic agents to the cells.

One of skill in the art can determine the appropriate amount based onthe above factors. The conjugate may be administered initially in asuitable amount that may be adjusted as required, depending on theclinical response of the subject. The effective amount of conjugate canbe determined empirically and depends on the maximal amount of theconjugate that can be administered safely. However, the amount ofconjugate administered is preferably the minimal amount that producesthe desired result.

Therefore, there is provided a pharmaceutical composition comprising aflavonoid conjugate hydrogel as described herein. The pharmaceuticalcomposition may further include a pharmaceutically acceptable diluent orcarrier. The pharmaceutical composition may routinely containpharmaceutically acceptable concentration of salts, buffering agents,preservatives and various compatible carriers. For all forms ofdelivery, the flavonoid conjugate hydrogel may be formulated in aphysiological salt solution.

The proportion and identity of the pharmaceutically acceptable diluentor carrier is determined by the chosen route of administration,compatibility with biologically active proteins if appropriate, andstandard pharmaceutical practice.

The pharmaceutical composition can be prepared by known methods for thepreparation of pharmaceutically acceptable compositions suitable foradministration to subjects, such that an effective amount of theflavonoid conjugates and any additional active substance or substancesis combined in a mixture with a pharmaceutically acceptable vehicle.Suitable vehicles are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., USA 1985). On this basis, thecompositions may include the flavonoid conjugate hydrogel in associationwith one or more pharmaceutically acceptable vehicles or diluents, andcontained in buffer solutions with a suitable pH and iso-osmotic withphysiological fluids.

Under ordinary conditions of storage and use, such pharmaceuticalcompositions may contain a preservative to prevent the growth ofmicroorganisms, and that will maintain any biological activity of theflavonoid conjugate hydrogel. A person skilled in the art would know howto prepare suitable formulations. Conventional procedures andingredients for the selection and preparation of suitable formulationsare described, for example, in Remington's Pharmaceutical Sciences andin The United States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999. Alternatively, the flavonoid conjugate hydrogel maybe formulated at a time sufficiently close to use by mixing thecomponents, without the need for preservatives.

The term “cell” as used herein includes a single cell, a plurality ofcells or a population of cells where context permits, unless otherwisespecified. The cell may be an in vitro cell, including a cell explantedfrom a subject. The cell may be a cell grown in batch culture or intissue culture plates. Alternatively, the cell may be an in vivo cell ina subject. In some embodiments, the subject is a human subject.Similarly, reference to “cells” also includes reference to a single cellwhere context permits, unless otherwise specified.

The term “stem cell” as used herein refers to an undifferentiated cellthat is capable of indefinite cell renewal and differentiation into avariety of cell types or a precursor cell that is partiallydifferentiated along a particular cell lineage and for which furtherdifferentiation is restricted to cells of that particular lineage. Thestem cell may be any type of stem cell, including an embryonic stem cellor an adult stem cell, including for example a mesenchymal stem cell.

Also presently provided is a hydrogel for adhering a cell, comprisingconjugates of a hydrogel forming agent and a flavonoid wherein saidconjugates are enzymatically cross-linked for adhesion of the cell tothe hydrogel. In one embodiment, the hydrogel is a hydrogel formed bythe methods disclosed herein.

The present methods and hydrogels are further exemplified by way of thefollowing non-limiting examples.

EXAMPLES Example 1

Materials and Methods

Materials: Hyaluronic acid (HA, 90KDa) was kindly donated by ChissoCorporation (Tokyo, Japan).1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HCl)and N-hydroxysuccinimide (NHS) was purchased from Sigma-Aldrich(Singapore). Hydrogen peroxide (H₂O₂) was obtained from Lancaster.Horseradish peroxidase (HRP, 100 units/mg) was purchased from Wako PureChemical Industries (Japan). DMEM media was obtained from Sigma-Aldrich(Singapore). 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI), andcell culture supplements were obtained from Gibco (InvitrogenSingapore). Fetal bovine serum (FBS) was purchased from Hyclone(Research Instrument, Singapore). Penicillin and streptomycin wereobtained from JRH biosciences (Singapore). AlamarBlue® was purchasedfrom TREK Diagnostic Systems (England). Other general use chemicals werepurchased from Sigma-Aldrich (Singapore). BD BioCoat Matrigel™ invasionchambers and cell culture inserts were purchased from BD Bioscience(USA). Unless stated otherwise, all reagents and solvents were ofbiological grade, and were used without further purification.

Synthesis of HA-diethylaminoacetal (HA-AA) conjugate: HA (5 g, 12.5mmol, Mw 90K) was added to 500 ml deionized water and allowed todissolve by vigorous stirring. Aminoacetaldehyde diethyl acetal (1.19 g,9.0 mmol, Sigma-Aldrich), NHS (1.16 g, 14 mmol) and EDC HCl (2.395 g,12.5 mmol, Sigma-Aldrich) were added and the pH of the solution wasadjusted to 4.7. The resulting solution was stirred overnight underambient conditions. The solution was then adjusted to pH 7 with 10MNaOH. The synthesized product was first dialyzed against 100 mM NaClsolution for two days, followed by dialysis in 25% v/v ethanol indeionized water for two days, and deionized water for two days using adialysis tubing (Spectrapore®7 membrane, MWCO=3500). Purified HA-AA waslyophilized. The degree of substitution (the number ofdiethylaminoacetal molecules per 100 repeating units of HA) wascalculated from ¹H NMR measurement by comparing the ratio of therelative peak integrations of methyl proton of diethylaminoacetal (peakat 0.99-1.04 ppm) and methyl proton of HA (1.93 ppm). The degree ofsubstitution was 12. ¹H NMR (D₂O): δ 0.99-1.04 (m, 6H methyl of diethylacetal), 1.829 (3H, N-acetyl), 3.10-3.70 (14H from HA part and 4 H fromthe ethyl groups of AA, five broad signals), 4.10-4.40 (2H, HA protonsfrom the acetamide and acid group bearing carbons), 4.465 (2H, protonattached to the acetal carbon, disappears upon deprotection).

Synthesis of HA-EGCG conjugates: HA-AA (712 mg, 1.83 mmol HA units,0.216 mmol acetal units) was dissolved in deionized water (40 mL) anddegassed by bubbling nitrogen through the solution. In a separate flask,epigallocatechin gallate (EGCG, 1 g, 2.18 mmol, 10.09 equivalents withrespect to the acetal units) was added to 8 ml of degassed DMSO, andallowed to dissolve by stirring at room temperature. The DMSO solutionof EGCG was then mixed with the solution of HA-AA under nitrogenatmosphere, the pH of the solution was adjusted to pH 1 usingconcentrated HCl while bubbling with nitrogen. The reaction was allowedto occur at room temperature for 24 h. The resulting solution wasdialyzed against degassed deionized water for 3 days, followed bylyophilization.

Synthesis of enzymatically cross-linked HA-EGCG hydrogel: 1 μl of H₂O₂solution (1.42 mmol/1) and HRP solution (25 units/ml) at various volume(0 to 25 μl) were added sequentially to a well of 96-well cell cultureplate. To this 100 of HA-EGCG (20 mg/ml) Dulbecco's Modified Eagle'sMedium DMEM) solution containing 10% fetal bovine serum (FBS) was addedand stirred vigorously with a pipet tip. The enzyme mediated oxidationreaction of HA-EGCG was allowed to proceed for 24 h inside a cellculture hood. Enzymatically cross-linked hyaluronic acid-tyramine(HA-Tyr) hydrogel, gelatin-(hydroxyphenyl)propionic acid (Gtn-HPA)hydrogel, and HA-Tyr/Gtn-HPA hydrogel were employed as controls in thisstudy. HA-Tyr hydrogel and Gtn-HPA hydrogels were prepared as describedpreviously [4, 15, 16]. HA-Tyr/Gtn-HPA mixed hydrogel were prepared bysimple mixing of HA-Tyr and Gtn-HPA polymer at various mass ratio,followed by oxidative coupling of tyramine or (hydroxyphenyl)propionicacid moieties catalyzed by hydrogen peroxide (H₂O₂)

Cell culture, cell adhesion and cell image analysis: Human foreskinfibroblast cells (HFF-1), human fibrosarcoma (HT-1080), and humanhepatocelluar carcinoma cells (Hep G2) were purchased from American TypeCulture Collection (ATCC, USA). Cells were grown and maintained in DMEMsupplemented with 10% FBS, 2 mM L-glutamine, and 50 units/ml penicillinstreptomycin at 37° C. in a humidified 5% carbon dioxide incubator.Cells were seeded at a density of 2×10⁴ per well in 96-well platescoated with hydrogel and incubated for the indicated time period at 37°C. Non-adherent cells were removed by rinsing with PBS. The surface ofthe gel was examined after 24-144 h of incubation using an Olympus IX71light microscope attached to a video camera. Image analysis of projectedcell area was performed with Image-Pro® Plus (MediaCybernetics, USA).The projected cell area measurement was obtained by tracing cellboundaries, and this parameter is displayed as an average (±standarderror of mean).

Cell proliferation on the surface of hydrogels: Hep G2 and HFF-1 cellswere seeded at 2×10⁴ cells per well in 96-well plates coated with 100 μlHA-EGCG or HA-Try/Gtn-HPA (80:20, w/w) hydrogels each cross-linked by 1μl of H₂O₂ (1.42 mmol/1) and 25 μl HRP solution (25 units/ml). Theproliferations of cells were evaluated in terms of AlamarBlue® reductionusing AlamarBlue® assay. After 24-96 h incubation, spent media werereplaced with fresh complete media containing 10% AlamarBlue®, andcultured for another 4 h. The cells seeded on HA-Tyr/Gtn-HPA (80:20 w/w)hydrogel were used as control. Hydrogel substrates without any cellswere used as blank controls. The absorbance values were recorded on amicroplate reader (GENios Pro, Tecan, Austria) with wavelength at 570 nmand 600 nm.

DNA fragmentation assay: After 120 h of incubation, cells cultured onthe hydrogel surface were washed twice with phosphate-buffered saline(PBS) and then fixed by 4% paraformaldehye for 30 min. The cells wererinsed with PBS, followed by incubation in a 1 μg/ml DAPI nucleic acidstain for 30 min in the dark. The cells were rinsed again and observedby a fluorescence microscope (Olympus, Tokyo, Japan).

Cancer cell invasion assay: HA-EGCG hydrogels were prepared by adding 20μl of HA-EGCG solution on cell culture inserts with 8 μm pore size.HT-1080 were (2.5×10⁴ cells/well) added to the upper compartment of theinvasion chamber. Culture medium was added to the lower compartment ofthe invasion chamber. The chambers were incubated at 37° C. in ahumidified 5% carbon dioxide incubator and cells were allowed to migratefor 90 h. Matrigel™ coated (filter inserts with 8.0 μm pore size)invasion chambers were used as a control. After incubation, the membraneinserts were removed from the wells, cells on the upper side of themembrane were removed using cotton swabs. The membranes were fixed,stained, and mounted according to the manufacturer's instructions.

Animal study: Tumour growth inhibition of human breast BT474 cancer inMice was examined. Mice with human breast BT474 cancer were treated witheither a control phosphate buffered saline solution (PBS), a HA-EGCGhydrogel (formed using 2.5 μl of 25 units/ml of HRP), herceptin or aherceptin loaded HA-EGCG hydrogel. The HA-EGCG hydrogel and herceptinloaded HA-EGCG hydrogen were administered once subcutaneously. Theherceptin was administered twice weekly intraperitoneally.

Materials: HA (90 KDa) was kindly donated by Chisso Corporation (Tokyo,Japan). Diethoxyethyl amine (DA), N-hydroxysuccinimide (NHS),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),dimethyl sulfoxide (DMSO), xanthine, sodium chloride (NaCl) and catalasefrom bovine liver were from Sigma (Singapore). Di-sodium hydrogenphosphate dihydrate and sodium dihydrogen phosphate monohydrate wereobtained from Merck (Singapore). Horeseradish peroxidase was purchasedfrom was from Wako Pure Chemical Industries (Japan). Ethanol ispurchased from Fisher Scientific (Singapore). EGCG (>95% purity) waspurchased from Kurita Water Industries (Tokyo, Japan).

Synthesis of HA-DA conjugates: HA-DA conjugates were synthesized using astandard carbodiimide coupling method. Briefly, HA (5 g, 12.5 mmol) wasdissolved in 500 ml of distilled water. To this diethoxyethyl amine (DA)with different amounts (1.19 g, 8.93 mmol or 2.38 g, 17.8 mmol) wasadded, followed by NHS (1.16 g, 10.0 mmol) and EDC (2.40 g, 12.5 mmol)to initiate the conjugation reaction. As the reaction proceeded, the pHof the mixture was maintained at 4.7. The reaction mixture was stirredovernight at room temperature after which the pH was increased to 7.0.The solution was transferred to dialysis tubes with a molecular cut-offof 1000 Da. The tubes were dialyzed against 100 mM NaCl solution for 2days, a mixture of distilled water and ethanol (3:1) for 1 day anddistilled water for 1 day, successively. The purified solution waslyophilized to obtain the HA-DA conjugate (4.2 g).

Synthesis of HA-EGCG conjugates. HA-DA conjugates with differentsubstitution degrees (1 g) were dissolved in 57 ml of distilled water.The solution was then purged with nitrogen gas for 20 min. EGCG (25equivalents of molar concentration with respect to the DA units) wasdissolved in 23 ml of nitrogen purged DMSO and added to the solution ofHA-DA conjugate. The pH of the solution was adjusted to 1.0 usingconcentrated HCl. The mixture was stirred at room temperature for 24 hunder a nitrogen atmosphere. After which the solution was transferred todialysis tubes with a molecular cut-off of 3500 Da and dialyzed againstdistilled water under nitrogen atmosphere for 3 days. The purifiedsolution was lyophilized to obtain the HA-EGCG conjugate (0.87 g).

Preparation of HA-EGCG solution: For all experiments described below,HA-EGCG stock solution was prepared by dissolving the conjugates indistilled water at 25 mg/ml at room temperature using a magneticstirrer. Dissolution took about 25 min and the pH of the solution was2.5. Then the pH was brought to 6 using 2 M NaOH before diluting withsodium phosphate buffer (final ionic strength: 0.15 M) to the desiredconcentration and pH, depending on the gelation condition.

Formation of HA-EGCG hydrogels through air autoxidation: To form HA-EGCGhydrogel by air autoxidation, HA-EGCG solution was diluted to 17.5 mg/mlby adding sodium phosphate buffer with pH between 6.0 to 8. To determinethe gelation time, glass vials containing 0.25 ml of HA-EGCG were tiltedfrequently at 90 degrees for 5 sec, the time at which no obvious flowingmotion could be observed was recorded as the gelation time.

Acceleration of HA-EGCG hydrogel formation through air autoxidation byadding catalase: Stock solution of catalase was prepared by dissolvingthe enzyme in distilled water at 22.5 kU/ml. The final HA-EGCGconcentration was 17.5 mg/ml at pH 7.4 and the catalase concentrationranged from 0 to 4 kU/ml. The gelation time was determined as describedabove.

Measurement of H₂O₂ Production by HA-EGCG: The amount of H₂O₂ producedby HA-EGCG was determined using the PeroXOquant Quantitative PeroxideAssay Kits from Pierce. In order to determine the amount of H₂O₂produced during the dissolution process, a sample of the dissolvedconjugates was diluted 20-fold with distilled water, 20 μl of which wasadded to the well of a 96-wellplate follow by the addition of 200 μlworking reagent prepared according to manufacturer's protocol. TheHA-EGCG solution was then diluted to 10 mg/ml with phosphate buffer. Thefinal ionic strength of the mixture was 0.15 M and pH was 7.4. For thenext 35 min, 20 μl of the HA-EGCG solution was drawn out every 5 min anddiluted 10-fold with distilled water. 20 μl of the diluted sample wasthen added to a well of a 96-wellplate followed by 200 μl of workingreagents. After the last sample was collected and the working reagentadded, the plate was incubated for another 1 h before reading theabsorbance at 595 nm. The amount of H₂O₂ produced by HA-EGCG wasdetermined by comparing to a set of H₂O₂ standards.

Formation of HA-EGCG hydrogel by HRP-mediated crosslinking reaction: HRPstock solution was prepared in water at 6.25 U/ml. The final HA-EGCGconcentration was 17.5 mg/ml at pH 7.4 and the HRP concentration rangedfrom 0 to 0.13 U/ml. The gelation time was determined as describedabove.

Measurement of storage modulus (G′) of HA-EGCG Hydrogels: 500 ul of theHA-EGCG solution at different pH, or containing different concentrationof catalase or HRP, were placed on top of a glass plate. The glass platewas covered with a thin later of Parafilm to prevent the hydrogel fromsticking to the glass surface. A second plate, also Parafilm-coated, wasthen placed on top to sandwich the hydrogel within a 1.5 mm gap, forminga circular hydrogel slab with diameter of approximately 2 cm. The plateswere then wrapped with cling wrap to prevent evaporation and thehydrogels were allowed to form at 37° C. in a humidified environment for24 hrs. After which the hydrogels were removed and gently placed on thebottom plate of a plate-plate serrated sensor (PP20) in a HAKKERheoscope 1. The top plate was then lowered to a measurement gap of 0.9to 1 mm. The measurement parameters were performed in controlleddeformation mode of 0.1% at 0.1 Hz. Preliminary experiments were done toensure that the measurement parameters were within the linearviscoelastic range of the hydrogels. Each reading was an average of fourmeasurement cycles and the first four readings were averaged to give theG′.

Results

Cell adhesion on HA-EGCG: In order to investigate whether HA-EGCGhydrogel can provide anchorage for cell attachment, adhesion assays wereperformed by seeding cells onto the surface of hydrogel. Representativephotomicrographs of the HT-1080 cells cultured for 24 h on varioushydrogels are shown in FIG. 2. It is well known that cells do not attachon HA-based hydrogel because cells adhesion is prevented by thehydrophilicity of hyaluronic acid [17, 18]. Some studies have showedimproved cell attachment by conjugating RGD peptide, collagen or gelatinto HA-based hydrogels [19, 20]. In the present study, HA-Tyr hydrogeland HA-Tyr/Gtn-HPA (20:80 w/w) hydrogel were used as negative andpositive control, respectively (FIGS. 2c and d ). Cells were also seededonto a plastic plate (FIG. 2a ) and on a Gtn-HPA hyrdogels (FIG. 2b ) ascontrols. Cells attached to HA-Tyr/Gtn-HPA (80:20 w/w) hydrogel. It wasobserved that cells did adhere loosely to HA-Tyr, but never spread outand they could easily be detached by rinsing with PBS for assay. Theseresults were consistent with other hyaluronan system [17, 18].Interestingly, cells could be attached and uniformly spread out on thesurface of HA-EGCG prepared by enzyme-mediated oxidation (FIG. 2f ).However, similarly to the HA-Tyr hydrogel, cells only adhered loosely toHA-EGCG hydrogel prepared by oxygenic-oxidation (FIG. 2e ).

These results provide evidence that the presence of the EGCG domainconferred the cell binding affinity to the HA-EGCG hydrogel. Thedifferences in adhesiveness might be due to the difference in thecross-linked EGCGs structure. Under typical cell culture conditions,EGCG undergoes oxygenic-oxidation (known as auto-oxidation) to producesuperoxide radicals and EGCG radicals with unpaired electronsdelocalized around the B ring. A chain reaction is propagated by thereaction of superoxide with EGCG, generating EGCG dimers and H₂O₂ [9,21]. The dimerization of EGCG results in cross-linking of HA-EGCGyielding hydrogel formation. In contrast, in the presence of HRP, thepossible mechanistic pathway of enzymatic oxidation of HA-EGCG isproposed as follows: HRP catalyzes the decomposition of H₂O₂ at theexpense of aromatic proton donors, leading to the coupling of phenolswith H₂O₂ as an oxidant and resulting in the formation of oligomericcompounds consisting of phenylene and oxyphenylene units [22]. Thehydrogel produce from enzymatically cross-linked HA-EGCG can consist ofa complex mixture of EGCG dimers and EGCG oligomers. The HRPconcentration play a role in determining the ratio of EGCG oligomers todimers. The ratio of oligomers to dimers can be elevated by increasingthe HRP concentration. Enzymatic oxidation is overwhelmingly faster thanoxygenic oxidation and exhausting the supply of EGCG in enzymaticoligomerization can inhibit the oxygenic dimerization of EGCG. The ratioof EGCG oligomers to dimers can therefore be modulated by varying theHRP concentration. The results demonstrated that enzymatic cross-linkedHA-EGCG hydrogel allowed cells to adhere and provided better anchoragefor cell spreading compared to oxygenic-cross-linked HA-EGCG hydrogel.This data provided evidence that the strength of cell adhesion can bemanipulated by varying the HRP concentration which controls the ratio ofoligomers to dimers of EGCG produced during cross-linking.

Cell spreading responses: Cellular adhesion and integration with tissueis an important prerequisite for the design of tissue engineeringconstructs. HA-EGCG hydrogel has shown cellular adhesive properties asdescribed earlier. It was important to further examine cellular activityon HA-EGCG. Cell functions such as spreading and proliferation areanchorage-dependent, and cell shape is affected by the extent of cellspreading. Cell spreading can be readily quantified by measuring itsprojected cell area and this parameter can also be used as an indicatorfor cell viability and proliferation. For example, cells such asfibroblasts typically exhibit spindle-shape morphology, the intermediateprojected cell area is maximal with this morphology [23].

Quantification of the degree of dimerization or oligomerization in aHA-EGCG hydrogel remains a technical challenge, however, study of therelationship of HRP concentration and the physiological behaviour ofcells provides insight into the structural importance of HA-EGCG.HT-1080 cells and Hep G2 cells were cultured on HA-EGCG hydrogelscross-linked by various amounts of HRP and the cell spreading behaviorwas examined. It has been reported that cell spreading issubstrate-stiffness dependent [24, 25]. In order to minimize mechanicalstrength variation between hydrogels in this study,enzymatic-cross-linked hydrogel with similar gel stiffness were preparedby varying HRP concentration but fixing the H₂O₂ concentration.

Representative photomicrographs of HT-1080 and Hep G2 cells are shown inFIG. 3a and FIG. 3b respectively. Cell spreading was remarkablydependent on the amount of HRP used for HA-EGCG gel formation.Concentrations of 0 units/ml (FIG. 3a (i) and FIG. 3b (i)), 2.3 units/ml(FIG. 3a (ii) and FIG. 3b (ii)), 3.2 units/ml (FIG. 3a (iii) and FIG. 3b(iii)), 4.1 units/m (FIG. 3a (iv) and FIG. 3b (iv)), and 4.9 units/ml((FIG. 3a (v) and FIG. 3b (v)). The cell attachment was greater and thecells were more uniformly spread on enzymatically cross-linked HA-EGCGthan oxygenic-cross-linked HA-EGCG, implying cells responded todifferent structure of HA-EGCG. The images also showed the tendency ofcells to extend projections.

Cell spreading was further quantified by projected cell area measurementas illustrated in FIG. 4a-c . After 24 h incubation, the projected cellareas of HT-1080, Hep G2, and HFF-1 cells cultured on enzymaticallycross-linked hydrogel were larger than those cultured onoxygenic-cross-linked hydrogel. FIG. 4a demonstrates the trend ofprojected cell area of HT-1080 cells which increased with increasing HRPconcentration and became steady as the concentration of HRP reached 3.2unit/ml, where the projected cell area reached a maximum (similar tocells cultured on a plastic well). For the case of Hep G2 cells, theprojected cell area increased with increasing HRP concentration until itreached the maximum (FIG. 4b ). The systematic increase in the extent ofcell spreading could be due to increasing density of adhesive domainspresented on the hydrogel. HA-EGCG hydrogel cross-linked with higher HRPconcentration might present more adhesive domains than HA-EGCGcross-linked with lower HRP concentration.

After 96-144 h, a pronounced difference in projected cell area wasfound, where cells cultured on oxygenic-cross-linked HA-EGCG hydrogelstill remained in spherical shape, as compared to spread cells adheredto enzymatically-cross-linked HA-EGCG hydrogel. As revealed by theprojected cell area in FIG. 4c , HFF-1 cells remain viable and spread insimilar manner as cells cultured on plastic well-plate. It isinteresting to note that, on the other hand, the projected cell areas ofHT-1080 and Hep G2 cells decreased (FIGS. 4a & 4 b). The decrease in theextent of cell spreading could be due to a decrease in cell viability,where HA-EGCG exerts an anti-proliferative effect on cancer cells. Asshown in FIG. 4b , the decrease in Hep G2 projected cell area seems tobe HRP dependent. This data provides further evidence that cell adhesionto and bioactivity of HA-EGCG is related to the cross-linked structureof EGCG.

Cell proliferation and DNA fragmentation on HA-EGCG hydrogel: Withregard to biocompatibility concerns, HA-EGCG hydrogel should exhibitminimal cytotoxicity to normal cells. To investigate the cytotoxiceffect of HA-EGCG, the proliferation of HT-1080 and HFF-1 cells wereexamined by phase contrast microscopy and quantitatively using acolorimetric growth indicator based on detection of metabolic activity.As cells proliferate, innate metabolic activity results in a chemicalreduction of AlamarBlue®, thus causing a change in colour. The amount ofreduced AlamarBlue® was calculated based on the absorbance valuesobtained according to the manufacturer protocol.

Representative photomicrographs of the cell morphology for HT-1080, HepG2 and HFF-1 cultured on HA-EGCG hydrogels following incubation times of24 hours (a), 48 hours (b), 96 hours (c) and 144 hours (d) are shown inFIGS. 5, 6 and 7, respectively. The HT-1080 and Hep G2 cell numbers werefound to decrease in relation to incubation time on HA-EGCG hydrogel.HT-1080 and Hep G2 cells became more spherical at the same time,indicating that they became less viable after culturing on HA-EGCGhydrogel. On the other hand, it was observed that HFF-1 cells continuedto proliferate until confluence (FIG. 7d ). These observations areinherent in the quantification of cell spreading (in FIG. 4 a,b,c) andAlamarBlue® assay (in FIG. 8a-b ). FIG. 8a-b shows the metabolicactivity of HT-1080 and HFF-1 cells on HA-EGCG and the controlHA-Tyr/Gtn-HPA hydrogel. As shown in FIG. 8a , HT-1080 metabolicactivity was found to decrease in relation to incubation time on HA-EGCGhydrogel. In contrast, no significant decrease of metabolic activity wasfound for HFF-1 cells plated on HA-EGCG (FIG. 8b ).

The mechanism underlying the suppression in cancer proliferation byHA-EGCG is not well understood. In order to identify whetherproliferation suppression is due to cellular senescence or by HA-EGCGinduced apoptosis, a DNA fragmentation assay was performed, in whichapoptosis was visualized by DAPI staining. As shown in FIGS. 9a and 9b ,DNA fragmentation was observed in Hep G2 cells cultured on HA-EGCGhydrogel. This result indicated that HA-EGCG suppressed cancerproliferation through eliciting apoptosis, which in turn halts celldivision cycle. In addition, no DNA fragmentation was observed in HFF-1cells (data not shown).

HA-EGCG hydrogels did not induce any remarkable cytotoxicity againstnormal cells, but inhibited cell proliferation of carcinomas. EGCG hasbeen shown to inhibit proteasomes in cancer cells. EGCG can thereforeselectively inhibit cell proliferation and induce apoptosis in cancercells without adversely affecting normal cells [26]. The chemopreventiveactivity and chemotherapeutic effects of HA-EGCG is expected, becausetea polyphenols such as EGCG and theasinensin (EGCG oxide) are known tobe capable of inducing apoptosis, as well inhibiting tumour cell growthand tumorigenesis [9, 27, 28]. Fujimura et al., [29] identified that themetastasis-associated 67 kDa laminin receptor (67LR) confers EGCGresponsiveness to cancer cells and mediates the anticancer activity ofEGCG. The selectivity in the anti-proliferation effect of HA-EGCG oncancer cells could possibly be 67LR mediated. Data from this studysuggested that the potencies of EGCG may have been retained in theHA-EGCG hydrogel. Noda et al., [13] have shown that combined-treatmentof a chemotherapeutic agent with EGCG synergistically induced apoptosis.

Invasion assay: With the aim of establishing anti-metastatic constructscapable of preventing cancer cells migration, an invasion assay wasconducted to examine whether HA-EGCG hydrogels exert the anti-metastaticeffect on tumor invasion which is known of EGCG. A Matrigel™ invasionassay was used as control. Pictures of typical fields of stainedinvading cells are shown in FIG. 10. Cells that migrated throughMatrigel™ were stained (FIG. 10a ). In contrast, no invading cells werefound on the membrane underneath HA-EGCG hydrogel (FIG. 10b ), implyingpossible anti-metastatic effects of the HA-EGCG hydrogel on HT-1080migration. 67LR has been implicated in laminin-induced tumor cellattachment and migration, as well as tumor angiogenesis, invasion andmetastasis [29]. The anti-metastatic effect of HA-EGCG could be 67LRmediated or it might be attributed to interactions of EGCG with MMPgelatinases leading to down-regulation of proliferation [11].

Animal study: Sustained release of herceptin from the HA-EGCG hydrogeldemonstrated inhibition of tumour growth in mice with human breastcancer BT474 cells (FIG. 11).

Gelation time of auto-oxidation flavonoid conjugate hydrogel: Thegelation time of HA-EGCG hydrogels formed by air-autoxidation reducedfrom 12 h at pH 6 to 10 min at pH 8 (FIG. 12). The storage modulusincreased from 200 to 1000 Pa as the pH increased from 6 to 8. To speedup the air-oxidation process, the H₂O₂ generated by EGCGair-autoxidation was removed by the catalase, an enzyme that catalyzesthe decomposition of hydrogen peroxide to water and oxygen. At pH 7.4,the gelation time decreased from 42 to 23 min as catalase concentrationincreased from 0.162 to 4 kU/ml (FIG. 13). The storage modulus wasbetween 1000 to 1500 Pa.

Formation HRP-mediated crosslinking in the absence of exongenous H₂O₂:The amount of H₂O₂ generated by HA-EGCG during air-autoxidationincreased with time and was found to be in the micromolar range (FIG.14). At pH 7.4, the gelation time of HA-EGCG decreased from 50 to 9 minas the HRP concentration increased from 0 to 0.125 unit/ml (FIG. 15).The storage modulus was between 1000 to 1200 Pa.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

All technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art of thisinvention, unless defined otherwise.

Concentrations given in this specification, when given in terms ofpercentages, include weight/weight (w/w), weight/volume (w/v) andvolume/volume (v/v) percentages.

As used in this specification, the singular forms “a”, “an” and “the”include plural reference unless the context clearly dictates otherwise.As used in this specification, the terms “comprise”, “comprising”,“comprises” and other forms of these terms are intended in thenon-limiting inclusive sense, that is, to include particular recitedelements or components without excluding any other element or component.Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit ofthe invention.

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What is claimed is:
 1. A method for producing a hydrogel comprisingconjugates of a hydrogel-forming agent and a flavonoid, the methodcomprising: combining the conjugates in a solution, in the absence of anexogenously added peroxide and in the absence of a peroxidase; andcontrolling the gelation rate of the hydrogel by modifying the pH of thesolution to be in the range of from 6 to
 8. 2. The method of claim 1,further comprising adding catalase to the solution.
 3. The method ofclaim 1, wherein the flavonoid is a catechin-based flavonoid.
 4. Themethod of claim 3, wherein the flavonoid is epigallocatechin gallate. 5.The method of claim 1, wherein the hydrogel-forming agent is a polymer.6. The method of claim 5, wherein the hydrogel-forming agent ishyaluronic acid.
 7. The method of claim 1, wherein the concentration ofthe conjugates in the solution is from about 0.1 mg/mL, to about 500mg/mL.
 8. The method of claim 1, further comprising combining abioactive agent with the conjugates in the solution.
 9. The method ofclaim 8, wherein the bioactive agent is an anti-cancer agent.
 10. Themethod of claim 9, wherein the anti-cancer agent is herceptin.
 11. Amethod for producing a hydrogel comprising conjugates of ahydrogel-forming agent and a flavonoid, the method comprising: combiningthe conjugates and a peroxidase in a solution in the absence of anexogenously added peroxide; and controlling the gelation rate of thehydrogel by modifying the of he solution to be in the range of from 6 to8.
 12. The method of claim 11, wherein the flavonoid is a catechin-basedflavonoid.
 13. The method of claim 12, wherein the flavonoid isepigallocatechin gallate.
 14. The method of claim 11, wherein thehydrogel-forming agent is a polymer.
 15. The method of claim 14, whereinthe hydrogel-forming agent is hyaluronic acid.
 16. The method of claim11, wherein the concentration of the conjugates in the solution is fromabout 0.1 mg/mL to about 500 mg/mL.
 17. The method of claim 11, furthercomprising combining a bioactive agent with the conjugates in thesolution.
 18. The method of claim 17, wherein the bioactive agent is ananti-cancer agent.
 19. The method of claim 18, wherein the anti-canceragent is herceptin.